BD Accuri™ C6 Plus System

User’s Guide

For Research Use Only

23-18013-00
1/2016

Becton, Dickinson and Company BD Biosciences

BD Biosciences European Customer Support
2350 Qume Drive Tel +32.2.400.98.95
San Jose, CA 95131 USA Fax +32.2.401.70.94
[email protected]

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Copyrights
© 2016, Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced,
transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in
any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without
prior written permission from BD Biosciences.

The information in this guide is subject to change without notice. BD Biosciences reserves the right to change
its products and services at any time to incorporate the latest technological developments. Although this guide
has been prepared with every precaution to ensure accuracy, BD Biosciences assumes no liability for any errors
or omissions, nor for any damages resulting from the application or use of this information. BD Biosciences
welcomes customer input on corrections and suggestions for improvement.

Trademarks
Alexa Fluor® is a registered trademark of Life Technologies Corporation.

Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and
Carnegie Mellon University, and are made and sold under license from GE Healthcare only for research and in
vitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 800 Centennial
Avenue, Piscataway, NJ 08855-1327, USA.

Trademarks are the property of their respective owners.

© 2016 BD. BD, the BD Logo, and all other trademarks are property of Becton, Dickinson and Company.

Regulatory information
For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Class 1 Laser Product.

FCC information
WARNING: Changes or modifications to this unit not expressly approved by the party responsible for
compliance could void the user’s authority to operate the equipment.

NOTICE: This equipment has been tested and found to comply with the limits for a Class A digital device,
pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against
harmful interference when the equipment is operated in a commercial environment. This equipment generates,
uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction
manual, may cause harmful interference to radio communications. Operation of this equipment in a
residential area is likely to cause harmful interference in which case the user will be required to correct the
interference at his or her own expense. Shielded cables must be used with this unit to ensure compliance with
the Class A FCC limits. This Class A digital apparatus meets all requirements of the Canadian Interference-
Causing Equipment Regulations. Cet appareil numérique de la classe A respecte toutes les exigences du
Réglement sur le matériel brouilleur du Canada.

History

Revision Date Change made

23-18013-00 1/2016 Initial release

 Contents

Chapter 1: Introduction 7
About this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Additional documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Safety symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Chapter 2: About the system 13

BD Accuri C6 Plus system overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
BD Accuri C6 Plus cytometer overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
BD CSampler Plus overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
BD Accuri C6 Plus software overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Chapter 3: Startup and shutdown 35

Startup workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Filling the fluid bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Emptying the waste bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Starting up the system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Shutting down the system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Chapter 4: Quality Control 47

QC overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Running instrument QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Adjusting the bead regions and markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Viewing QC results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
iv BD Accuri C6 Plus System User’s Guide

Viewing Levey-Jennings plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Chapter 5: Manual data acquisition 67

Collect tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Setting acquisition parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Run settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Fluidics rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Running samples using the Collect tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Ending a data collection session . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Chapter 6: CSampler Plus data acquisition 87

Manual Collect tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Running samples using the Manual Collect tab . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Auto Collect tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Creating sample sets for Auto Collect experiments . . . . . . . . . . . . . . . . . . . . . . 103
Setting parameters for Auto Collect experiments . . . . . . . . . . . . . . . . . . . . . . . . 106
Running samples using the Auto Collect tab . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Ending a data acquisition session . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Chapter 7: Data analysis 119

Analyzing sample data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Setting up analysis plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Creating an overlay histogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Statistics tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Previewing a plot in the Statistics tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Adjusting peak position with VirtualGain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Copying analysis data into other applications . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Analyzing batches of samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
 Contents v

Chapter 8: Plots and gates 155

Plots and gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Creating a new plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Creating and applying gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
Creating nested gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Modifying regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Modifying plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Zooming a plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Copying and printing plot data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Chapter 9: Managing data 183

Managing files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Workspace files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
Creating a workspace template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Exporting and importing data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

Chapter 10: Maintenance 193

Maintenance schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Cleaning the outside of the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Backflushing the SIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Performing a SIP clean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Cleaning the fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Running an extended flow cell clean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Cleaning the fluid bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Replacing the fluid bottle filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Replacing the in-line sheath filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Replacing the peristaltic pump tubing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Purging the fluid sensor lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Unclogging the SIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Collecting precise volume measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Aligning the CSampler Plus after a collision . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
 Restarting the system after storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Chapter 11: Troubleshooting 221

Troubleshooting overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Hardware troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Software troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Acquisition troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
QC troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Chapter 12: Selectable Lasers Module 235

Selectable lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
Annotating selected laser configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Optical filter placement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Selectable laser application examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

Chapter 13: User Tracking Module 249

Tracking user activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
Managing user accounts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Monitoring user activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

Chapter 14: Remote Interfacing Module 259

Remote Interfacing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
Implemented commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
Example usage of remote interfacing commands . . . . . . . . . . . . . . . . . . . . . . . . 265

Chapter 15: FCS keywords 267

FCS keywords . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

Chapter 16: Technical specifications 273

System specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

Index 279
 1
Introduction
This chapter covers the following topics:

• About this guide (page 8)

• Additional documentation (page 9)
• Safety symbols (page 10)
• Technical support (page 11)
8 BD Accuri C6 Plus System User’s Guide

About this guide

Introduction This topic describes the information that is available in this guide.

In this guide This guide provides information for setting up and running the
BD Accuri™ C6 Plus system during a typical workflow, whether
you are acquiring samples manually or using a BD CSampler™
Plus accessory.

The guide includes:

• An overview of the BD Accuri C6 Plus system

• Introductory information about system hardware and
components
• Instructions on setting up and maintaining the system
• Instructions on performing daily instrument QC
• Information about BD Accuri C6 Plus software and
BD CSampler Plus software features, and how to use the
software to set up and run the cytometer
• Instructions on acquiring samples, with and without a
BD CSampler Plus
• Information on analyzing data using the software
 Chapter 1: Introduction 9

Additional documentation
Introduction This topic describes additional documentation available for the
BD Accuri C6 Plus system.

Documents The following table lists the available documents for the BD Accuri
C6 Plus system.

Document Description
BD Accuri™ C6 Plus Provides the site requirements. Read this
System Site Preparation guide before the system is installed.
Guide

BD Accuri™ C6 Plus Provides safety guidance and system

System Safety and limitations. Read this guide before
Limitations Guide running the system.

BD™ CS&T RUO Beads Provides instructions on preparing the

data sheet BD CS&T RUO beads for quality control.

BD Accuri™ C6 Plus A Microsoft Excel® file that provides

Compensation Calculator instructions on calculating compensation
values for any four-color experiment.

More information • About this guide (page 8)

• Safety symbols (page 10)
• Technical support (page 11)
10 BD Accuri C6 Plus System User’s Guide

Safety symbols
Introduction This topic describes the safety symbols used in this guide. For a
complete description of all safety hazards, see the BD Accuri C6
Plus System Safety and Limitations Guide.

Safety symbols The following table describes the symbols used in this guide.

Symbol Meaning
Caution. Indicates the need for the user to consult the
system user’s guide for important cautionary information
such as warnings and precautions that cannot, for a
variety of reasons, be presented on the device itself.

Biological hazard

Electrical hazard

Laser hazard

More information • Additional documentation (page 9)

 Chapter 1: Introduction 11

Technical support
Introduction This section describes how to get technical support.

Contacting BD To contact BD technical support:

technical support If assistance is required, contact your local BD Biosciences
technical support representative or supplier. Visit our website,
bdbiosciences.com, for up-to-date contact information.

When contacting BD Biosciences, have the following information

available:

• Product name, part number, and serial number

• Software application and version number
• Any error messages
• Details of recent system performance

Contacting Dell™ To contact Dell technical support:

technical support If you purchased the Dell computer through BD Biosciences, it
comes with a 3-year warranty. For technical assistance with your
Dell computer, visit www.dell.com/supportcontacts/.

When contacting Dell, have the following information available:

• Your name and contact information

• Service tag number
• Summary of the problem
• Troubleshooting steps taken
• Any error codes from diagnostics
• System location

More information • About this guide (page 8)

12 BD Accuri C6 Plus System User’s Guide
 2
About the system
This chapter covers the following topics:

• BD Accuri C6 Plus system overview (page 14)

• BD Accuri C6 Plus cytometer overview (page 16)
• BD CSampler Plus overview (page 24)
• BD Accuri C6 Plus software overview (page 28)
14 BD Accuri C6 Plus System User’s Guide

BD Accuri C6 Plus system overview

Introduction This topic describes the BD Accuri C6 Plus system and
components.

C6 Plus system The C6 Plus system includes the BD Accuri C6 Plus flow
cytometer, an all-in-one desktop workstation running BD Accuri
C6 Plus software (or BD CSampler Plus software) for acquisition
and analysis, and the optional BD CSampler Plus. The system also
includes BD™ CS&T RUO beads for instrument QC. An optional
barcode reader can be attached to a USB port on the computer.

C6 Plus cytometer The two-laser, six-parameter flow cytometer is composed of

fluidics, optics, and electronics systems.

• The fluidics system consists of peristaltic pumps providing a

non-pressurized, “push/pull” fluid system.
• Clustered in a pie configuration around the flow cell, the
optical detectors maximize light collection.
• The electronics system provides up to 7 decades of dynamic
range, allowing for fixed detector voltages.
 Chapter 2: About the system 15

Workstation and The system comes with a dedicated USB-compatible, all-in-one

software desktop workstation with keyboard and mouse. The workstation
runs BD Accuri C6 Plus software (or BD CSampler Plus software),
which is used to control the cytometer, acquire samples, and
generate results.

Note: While BD Accuri software is running, the computer will not

enter sleep mode. Do not change the power options or force the
computer to go to sleep while the software is running.

Reagents BD CS&T RUO beads are used to check and monitor the
cytometer performance. We recommend running instrument QC
daily, ensuring that the test passes before running samples.

The BD Accuri C6 Plus system works with many existing reagents

and protocols. Use the pre-defined templates with the
corresponding reagent kit. Template can be downloaded from
bdbiosciences.com. For instructions on preparing samples, see the
specific reagent kit package insert or data sheet.

CSampler Plus The BD CSampler Plus is an optional accessory that saves you time
option and effort by automating the sample loading step. Place a 24-tube
rack of standard 12 x 75-mm tubes or a 48- or 96-well microplate
on the CSampler Plus and select RUN. The CSampler Plus
automatically agitates the tubes to keep the cells in suspension,
then loads each tube for acquisition. Use the software to program
sample injection probe (SIP) cleaning and rinsing during the run.

More information • BD Accuri C6 Plus cytometer overview (page 16)

• BD Accuri C6 Plus system overview (page 14)
16 BD Accuri C6 Plus System User’s Guide

BD Accuri C6 Plus cytometer overview

Introduction This topic describes the cytometer and its major components.

Main components The C6 Plus cytometer is composed of fluidics, optics, and

electronics subsystems that work together to analyze cells.

Outside of The following figures show the front and back of the cytometer
cytometer and locations of the indicator lights, power button, sample
injection probe (SIP), and connectors.

Front

1 5
2

3
 Chapter 2: About the system 17

The following table describes the components on the front of the

cytometer.

No. Item Description

1 Event indicator Blinks as events pass in front of the laser. The
higher the event rate, the faster it blinks.

2 Power-on Illuminates solid blue to indicate that the

indicator cytometer is on and ready to use. Blinks
during startup and shutdown.

3 Power button Used to turn on and off the cytometer.

4 SIP Sample injection probe. Used to pull sample

from the sample tube to the flow cell.

5 Sample stage Used to hold the sample tube in place. Height

adjusts to accommodate 12 x 75-mm tubes
and BD Trucount™ tubes.
18 BD Accuri C6 Plus System User’s Guide

Back

The following table describes the components on the back of the

cytometer.

No. Item Description

1 Power connector Connects the AC power cord to the
instrument.

2 CSampler Plus Used only to connect the BD CSampler

connector Plus.

3 USB port Used to connect the computer workstation.

4 Fluidics harness Mesh outer tubing that contains fluid lines

connecting four fluid bottles to the internal
fluidics system.
Do not disconnect the harness from the
back of the instrument.
 Chapter 2: About the system 19

Optical The following figure shows the optical components.

components

4
20 BD Accuri C6 Plus System User’s Guide

The following table describes the optical components.

No. Component Description

1 Blue laser 488 nm

2 Red laser 640 nm

3 Optical assembly (four  FL1 533/30 nm

filters surround the flow  FL2 585/40 nm
cell and are outlined in
red)  FL3 >670 nm
 FL4 675/25 nm

4 Flow cell (in center of Capillary where the laser intersects

four filters) the sample stream

Fluidics The C6 Plus cytometer is a non-pressurized system. If necessary,

components any of the bottles can be opened while the cytometer is powered
on. However, we recommend waiting to fill the fluid bottles until
the instrument is idle.

During operation, the software will notify you if the fluid level for
a fluid bottle is getting low or the waste bottle is getting full.
Always attend to the fluid bottles when instructed.

The following figure shows the fluidics components. The plastic

storage bin located inside the instrument is not shown.
 Chapter 2: About the system 21

5 6

3
8
4

The following table describes the fluidics components.

No. Component Description

1 Sheath bottle 2-L bottle for 0.2-µm filtered deionized
(blue) (DI) water with BD™ Sheath Additive

2 Waste bottle (red) 2-L bottle to collect waste

3 Detergent solution 250-mL bottle for BD™ Detergent

bottle (green) Solution Concentrate

4 BD FACSClean 250-mL bottle for BD™ FACSClean

bottle (yellow) solution
22 BD Accuri C6 Plus System User’s Guide

No. Component Description

5 In-line sheath filter In-line filter to filter the sheath fluid.
Additionally, three disk filters (located in
the fluid bottles) filter the fluid in each
bottle (sheath, BD FACSClean, and
detergent solution).

6 Sheath pump Peristaltic pump that moves sheath fluid

from the sheath bottle through the system.

7 Waste pump Peristaltic pump that moves fluid to the

waste bottle.

8 SIP Sample injection probe. Pulls sample from

the sample tube to the flow cell. Shown in
figure in Outside of cytometer (page 16).

Loading a tube To manually load a tube:

1. Grasp the bottom of the sample stage between your thumb and
index finger and push it back.

2. Place the tube over the SIP.

 Chapter 2: About the system 23

3. While holding the tube, pull the sample stage forward to

support the tube.

Adjusting the Note: This procedure applies to manual acquisition only. If you
height of the are using a CSampler Plus for acquisition, the sample stage was
sample stage removed to accommodate for the CSampler Plus.

The bottom of the sample stage contains a metal platform that can
be adjusted to two different positions. It can be raised or lowered
to accommodate both standard 12 x 75-mm tubes and
BD Trucount™ tubes, respectively.

When loading BD Trucount tubes you can lower the platform to

prevent the SIP from hitting the retainer at the bottom of the tube.
You can also run 12 x 75-mm tubes in the lowered position. Or,
you can raise the sample stage platform when loading standard
12 x 75-mm tubes to allow the SIP to reach the bottom of the tube.

To adjust the height of the metal platform in the sample stage:

1. Push up on the underside of the metal platform in the sample
stage to raise the platform. Then, using your other hand,
swivel the platform 90° to raise/lower it.
24 BD Accuri C6 Plus System User’s Guide

Each 90° turn will raise or lower the platform.

BD CSampler Plus overview

Introduction This topic describes the BD CSampler Plus and how to use it to
automatically load samples.

About the The BD CSampler Plus is an optional sample loading accessory

CSampler Plus that agitates and delivers tubes to the C6 Plus cytometer for sample
acquisition. The CSampler Plus can be included as an option on a
new system, or it can be ordered and installed at a later time.

The CSampler Plus holds a custom rack of 24 standard 12 x 75-

mm tubes, 48- or 96-well plates, or BD Trucount tubes. One rack
is supplied with the CSampler Plus. Do not use any racks other
than the one supplied for this CSampler Plus.

Three fixed tube locations at the back of the CSampler Plus tray
allow you to load tubes for cleaning, rinsing, and backflushing the
SIP.
 Chapter 2: About the system 25

Loading a rack/ Always use the Eject Plate/Rack and Load Plate/Rack buttons in
plate the software to move the CSampler Plus. Never move the
CSampler Plus by hand.

Caution: Moving parts! Keep your hands away from the

CSampler Plus when it is moving. Never move the
CSampler Plus by hand.
To load a rack:
1. If necessary, click Eject Plate on the Collect or Manual Collect
tab, or click Eject Rack on the Instrument QC tab.

2. Place the 24-tube rack or plate on the CSampler Plus.

26 BD Accuri C6 Plus System User’s Guide

The rack or plate fits one way on the tray, ensuring that
position A1 is in the upper-left corner.

3. Press RUN on the Collect tab or Instrument QC tab to load

the rack or plate and begin acquisition.

Note: If you simply need to change the cleaning tubes, click

Eject Plate and replace the tubes, followed by Load Plate.

The CSampler Plus positions the selected tube under the SIP
for acquisition. A plate map on the screen allows you to see the
tube that is being acquired. The location flashes blue and is
outlined in red.

Cleaning tube The CSampler Plus is equipped with three fixed tube locations to
locations support the system cleaning functions. Check and replace these
tubes after system startup, after every run, and as needed.
 Chapter 2: About the system 27

Each location is designated by a symbol. The following table

describes the details of each location.

Location Description
Triangle ( ) Place a tube containing 2 mL of BD FACSClean in
this location. Used for the first tube in a SIP Clean.

Circle ( ) Place a tube containing 2 mL of DI water in this

location. Used for the second tube in a SIP Clean.

Square ( ) Place a tube containing 2 mL of DI water in this

location. Used at startup and shutdown, during a
backflush, and for a SIP Rinse. Also used for
parking the SIP at shutdown.

Recommendations This list provides recommendations to follow when using a

when using the CSampler Plus:
CSampler Plus
• Keep fresh cleaning tubes in the three fixed locations during
operation. For optimal performance, change the tubes after
startup, after every run, and as needed. If left unattended, the
tube in the square location could overflow.
• Sample tubes should not contain more than 2.5 mL of sample
to avoid spillover during agitation. Cleaning tubes in the three
fixed locations should not contain more than 2 mL of liquid.
• Use the software to move the CSampler Plus. Do not try to
move it with your hands.
• Keep your hands and objects out of the path of the CSampler
Plus.
• Ensure the SIP is parked in water at shutdown.

More information • CSampler Plus data acquisition (page 87)

• Aligning the CSampler Plus after a collision (page 217)
28 BD Accuri C6 Plus System User’s Guide

BD Accuri C6 Plus software overview

Introduction This topic describes BD Accuri C6 Plus software and BD CSampler
Plus software.

About the software BD Accuri C6 Plus software allows you to control the BD Accuri
C6 Plus flow cytometer system to acquire data, generate statistics,
and analyze results.

The software provides the following features:

• Tabbed views for collection, analysis, and statistics

• Quality control (QC) module that allows you to check and
monitor the instrument’s performance over time
• Plots that display more than six decades of dynamic range
• Color compensation at any time
• Drag and drop plots
• Batch analysis of sample data
• Export of files in FCS 3.1 format
• Seamless importation into FCS Express™ and other third-
party software
• Color gating
• Live gating

About BD CSampler BD CSampler Plus software allows you to control the BD Accuri
Plus software C6 Plus flow cytometer system and the BD CSampler Plus to
acquire data, generate statistics, and analyze results.

The software provides all the same features as the non-CSampler

Plus version. Additionally, it:

• Automatically acquires samples from a 48- or 96-well plate or

24-tube rack
• Ejects and loads the CSampler Plus tray
• Agitates the plate/tube rack and performs a SIP rinse between
samples
 Chapter 2: About the system 29

Software The main C6 Plus software window is called the workspace. The
workspace workspace contains controls and displays that provide access to all
the functions for acquiring and analyzing data.

The workspace is organized into four separate tabs. Additionally,

the Instrument QC button opens a separate QC window.

• Collect. Contains controls for setting up data collection and

acquiring data. See Collect tab (page 68) for more
information.

• Analyze. Contains controls for analyzing data. See Analyzing

sample data (page 120) for more information.

• Statistics. Displays statistical information. See Statistics tab

(page 130) for more information.
30 BD Accuri C6 Plus System User’s Guide

• Batch Analysis. Contains controls for analyzing batches of

sample data. See Analyzing batches of samples (page 142) for
more information.

CSampler Plus The main BD CSampler Plus software window is called the
workspace workspace. The workspace contains controls and displays that
provide access to all functions required for data acquisition and
analysis.

The BD CSampler Plus software workspace is organized into five

separate tabs. Additionally, the Instrument QC button opens a
separate QC window.

• Manual Collect. Contains controls for setting up data

collection and acquiring data in any order. See Manual Collect
tab (page 88) for more information.
 Chapter 2: About the system 31

• Auto Collect. Contains controls for automatically collecting

data from several wells in order (horizontally or vertically),
starting at a designated well. See Auto Collect tab (page 99)
for more information.

• Analyze. Contains controls for analyzing data. Allows you to

analyze multiple samples simultaneously. See Analyzing
sample data (page 120) for more information.

• Statistics. Displays statistical information. See Statistics tab

(page 130) for more information.

• Batch Analysis. Contains controls for analyzing batches of

sample data. See Analyzing batches of samples (page 142) for
more information.

Software menus This topic contains tables that list and describe the menu options
and options for BD Accuri C6 Plus software and BD CSampler Plus software.

File menu

Menu options Description

Open workspace or Opens a previously saved workspace file or template. Only one
template workspace can be open at a time.

New workspace Opens a new, blank workspace. Replaces any previously open
workspace.

Save Saves the open workspace under the current name. If the file has
not already been named, you will be prompted to name the file.

Save workspace as Saves the open workspace under a new name.

Save template as Creates a template from the currently open workspace. Markers,
regions, gates, parameter names, sample names, and user-defined
compensation settings are saved without any data points.

Auto-save Data Settings Allows you to enable or disable the auto-save feature.

Import FCS File Imports an FCS file previously exported from another file to the
currently open workspace. Only FCS files created using
BD Accuri C6 Plus software and BD Accuri™ C6 software can
be imported into the software.
32 BD Accuri C6 Plus System User’s Guide

Menu options Description

Export FCS File Exports and saves the currently selected data well as an FCS 3.1
file to a specified folder.
Exported files are compatible with off-line analysis programs
such as FCS Express, FlowJo™, and WinList™.

Export ALL Samples as Saves all of the data wells as individual FCS 3.1 files in the folder
FCS FCS Exports on the computer desktop. Exported files are
compatible with off-line analysis programs such as FCS Express,
FlowJo, and WinList.

Export Plot Data as CSV Saves an individual file in CSV format for further analysis in
spreadsheet programs. All data for every event in the selected
plot is exported.

Export Sample Settings Exports sample settings as a CSV file for viewing data in a
spreadsheet. Sample settings include acquisition criteria, sample
names, parameter names, and compensation values. Available in
BD CSampler Plus software only.

Preferences Allows you to select between using your own custom

compensation settings or using the compensation settings
obtained from instrument QC.

Set Plot Drag and Drop Allows plot format to be toggled between .png (low resolution)
Format or .eps (high resolution).

Print Selected Items Prints selected plots and associated statistics.

Quit Closes the software.

Edit menu

Menu options Description

Undo Undoes the last action that was performed in the software. You
can undo up to the last five actions. Not all actions can be
undone.

Redo Reverses an Undo action.

 Chapter 2: About the system 33

Menu options Description

Copy Copies a marker or region from a plot, or statistics from the
tables to the clipboard.

Paste Pastes copied markers and regions into new plots.

Rename Parameters/Color Allows you to rename individual parameters for either the
Compensation current sample or all samples at the same time. Also allows you
to view and/or change the color compensation settings.

Display menu

Menu options Description

Events Display Settings Opens a dialog that allows you to change the number of events
displayed in all plots.

Auto-Select Next Well Opens a dialog to configure whether the software automatically
selects wells vertically or horizontally, or not at all at the
completion of each sample acquisition.

Remove All VirtualGain™ Removes all VirtualGain settings from the entire workspace.
VirtualGain settings are removed from all wells of data.

Hide/Show Median Hides/shows the median statistics in the Statistics table.

Statistics

Instrument menu

Menu options Description

Set threshold Opens the Threshold dialog for selecting the trigger channel,
setting the primary threshold value, and setting an optional
secondary threshold.

Set compensation Opens the Compensation Settings dialog for subtracting

fluorescence spillover.

Run Clean Fluidics Runs a cleaning fluid cycle.

Run Backflush cycle Runs the backflush cycle to clean the SIP and remove clogs at the
base of the SIP.

Extended Flow Cell Clean Cleans the flow cell for an extended time.
34 BD Accuri C6 Plus System User’s Guide

Menu options Description

Align CSampler Plus Aligns the BD CSampler Plus arm to the SIP. Available for
BD CSampler Plus software only.

Auto-adjust volume Adjusts the fluidics to ensure that the cytometer accurately
settings reports the correct volume of sample.

Update firmware Used to update the firmware. Use only when directed by a BD
representative.

Remote Interfacing Optional feature available with automation key. Allows control
Module of the cytometer from a remote location.

About menu

Menu options Description

About BD Accuri C6 Plus Opens a dialog that displays the version of software and
software BD Technical Support contact information.

Technical Support Opens a dialog that displays information about the current
Information version of C6 Plus software (or CSampler Plus software) and the
cytometer. Each time an activation key is used to install a new C6
Plus software component, the dialog is updated to reflect the
change.

Users Appears when the optional User Tracking option is installed.

User tracking allows you to add, delete, or modify user accounts.
See Tracking user activity (page 250) for more information.

Create Cytometer Log Creates and opens a Microsoft Notepad document with
cytometer- and software-specific information for use by a
technical support representative. The file is saved to
C:\Cytometer Support Files.
 3
Startup and shutdown
This chapter covers the following topics:

• Startup workflow (page 36)

• Filling the fluid bottles (page 36)
• Emptying the waste bottle (page 39)
• Starting up the system (page 41)
• Shutting down the system (page 43)
36 BD Accuri C6 Plus System User’s Guide

Startup workflow
Introduction This topic describes the workflow for daily system startup.

Workflow The following table lists the tasks that should be performed each
day to start up the system and prepare it for running samples.

Step See
1 Filling the fluid bottles (page 36)

2 Emptying the waste bottle (page 39)

3 Starting up the system (page 41)

More information • BD Accuri C6 Plus cytometer overview (page 16)

Filling the fluid bottles

Introduction This topic describes how to fill the sheath, cleaning solution, and
detergent solution bottles.

About the fluid Visually check all the bottles at the start of each day and fill the
bottles sheath, BD FACSClean, and detergent solution bottles, as needed.

The cytometer is a non-pressurized system. If necessary, any of the

bottles can be opened while the cytometer is powered on.
However, avoid filling the fluids during startup, shutdown,
acquisition, and the cleaning cycles.
 Chapter 3: Startup and shutdown 37

The software displays a message when a fluid bottle needs

attention.

Caution: Always fill or empty the fluid bottle when the

message appears. Allowing the bottle to run dry can
introduce air in the lines and lead to problems with the
fluidics system.

Required materials The following fluids are used with the system:

Bottle name Fluid solution

Sheath 0.2-µm filtered DI water with BD Sheath
Additive

BD FACSClean BD FACSClean cleaning solution

Detergent Solution BD Detergent Solution Concentrate

Caution: Corrosive! BD Sheath Additive and

BD FACSClean contain chemicals which may be harmful
and can cause skin and eye irritation. Use universal
precautions when handling.

Before you begin Prepare the following solutions:

• Working solution of the BD Detergent Solution Concentrate

Add 3 mL of BD Detergent Solution Concentrate to 197 mL of
filtered DI water. Use the working solution within 2 weeks.

• Sheath fluid
Add one bottle (5 mL) of BD Sheath Additive to 1 liter of 0.2-
µm filtered DI water.
38 BD Accuri C6 Plus System User’s Guide

Procedure To fill the fluid bottles:

1. Disconnect each color-coded line from the top of the bottle by
squeezing the quick-connect fitting and pulling the tubing
connector out of the fitting.

2. Remove the cap from each bottle.

3. Fill each bottle with the appropriate fluid. Add:

• 2 L of filtered DI water with BD Sheath Additive to the

sheath fluid bottle (blue)
• 250 mL of BD FACSClean solution to the BD FACSClean
bottle (yellow)
• 250 mL of BD Detergent Solution Concentrate working
solution to the Detergent Solution bottle (green)
4. Replace the caps on the fluid bottles.

5. Snap the color-coded tubing back into place by pushing firmly

until you hear a click.

Next step Emptying the waste bottle (page 39)

More information • Fluidics components (page 20)

• Startup workflow (page 36)
 Chapter 3: Startup and shutdown 39

• Emptying the waste bottle (page 39)

• Replacing the fluid bottle filters (page 203)

Emptying the waste bottle

Introduction This topic describes how to safely empty the waste bottle.

About the waste Empty the waste bottle daily or when prompted by the software to
bottle prevent spillover and possible biological safety risk.

The cytometer is a non-pressurized system. If necessary, you can

empty the waste while the cytometer is powered on.

Caution: Biohazard! Always use precautions when

handling biological specimens or instruments that enter
in contact with specimens. Wear suitable protective
clothing, eyewear, and gloves. Dispose of waste using
proper precautions and in accordance with local
regulations.

Required materials • 200 mL of undiluted bleach

Procedure To empty the waste bottle:

1. Disconnect both connectors—waste tubing and fluid sensor—
from the top of the waste bottle by squeezing the quick-
40 BD Accuri C6 Plus System User’s Guide

connect fittings and pulling the tubing connectors out of the

fittings.

2. Carefully remove the cap.

3. Dispose of waste using proper precautions and according to

local, state, and country biohazard handling regulations.

4. Add approximately 200 mL of undiluted bleach to the bottle.

5. Replace the cap.

6. Snap the tubing back into place by pushing each connector

firmly into its color-coded fitting until you hear a click.

Next step Starting up the system (page 41)

More information • Fluidics components (page 20)

• Startup workflow (page 36)
• Filling the fluid bottles (page 36)
• Replacing the fluid bottle filters (page 203)
 Chapter 3: Startup and shutdown 41

Starting up the system

Introduction This topic describes how to turn on the cytometer and start
BD Accuri C6 Plus software. You can turn on the cytometer and
computer in any order.

Before starting the Check the fluid levels in all bottles.

cytometer
• Check the sheath bottle to ensure there is enough sheath fluid
for a run.
• Check the BD FACSClean and detergent solution bottles.
• Check the waste bottle to ensure there is adequate capacity.
If necessary, fill the fluid bottles and empty the waste. See Filling
the fluid bottles (page 36) and Emptying the waste bottle (page 39)
for information.

About startup During startup, the power indicator flashes blue and the traffic
light turns yellow while the laser warms up and the cytometer
flushes the fluid lines with fresh sheath fluid. This process takes
approximately 15 minutes.

If the power and indicator lights both flash, see Hardware

troubleshooting (page 222).

Note: Do not open the lid of the cytometer during the startup
process. Opening the lid interrupts the laser warm-up and extends
the time before samples can be acquired.

Required materials • For systems without a CSampler Plus. 2 mL of DI water

• For systems with a CSampler Plus. 2 mL of DI water (x2) and

2 mL of BD FACSClean solution
42 BD Accuri C6 Plus System User’s Guide

Opening BD Accuri To open the software:

C6 Plus software 1. Turn on the power to the computer.

2. Double-click the BD Accuri C6 Plus software icon (or

BD CSampler Plus software icon) on the computer desktop.
The software opens a new, blank workspace.

Starting the To start up the cytometer when running manually:

cytometer when 1. Ensure a tube containing at least 2 mL of DI water is loaded
running manually on the SIP. If necessary:

a. Push the sample stage back.

b. Place the tube of water over the SIP.
c. While holding the tube, pull the sample stage forward to
support the tube.
2. Press the power button on the front of the cytometer to turn it
on.

Once the fluid lines are flushed, the traffic light turns green
and the software displays the message Cytometer is connected
and ready. The power indicator on the front of the cytometer
turns solid blue.

Starting the To start up the cytometer when using the CSampler Plus:
cytometer when 1. Press the power button on the front of the cytometer to turn it
using the CSampler on.
Plus
Once the lasers are warmed up and the fluid lines are flushed,
the traffic light turns green and the software displays the
message Cytometer is connected and ready. The power
indicator on the front of the cytometer turns solid blue.
 Chapter 3: Startup and shutdown 43

2. Click Eject Plate and load the following fresh cleaning tubes in
the designated fixed locations on the CSampler Plus tray:

Caution! Do not add more than 2 mL of fluid to the

tubes. Do not overfill.

• A tube with 2 mL of BD FACSClean in the triangle ( )

• A tube with 2 mL of DI water in the circle ( )
• A tube with 2 mL of DI water in the square ( )
3. Click Load Plate.

More information • Filling the fluid bottles (page 36)

• Emptying the waste bottle (page 39)
• BD Accuri C6 Plus cytometer overview (page 16)
• BD CSampler Plus overview (page 24)
• Shutting down the system (page 43)
• Cleaning the fluidics (page 200)
• Performing a SIP clean (page 198)

Shutting down the system

Introduction This topic describes how to exit the software and turn off the
cytometer. You can turn off the cytometer and computer in any
order.

About shutdown When you turn off the power to the cytometer, the Clean Fluidics
cycle is run automatically. The cycle takes about 13 minutes to
complete. See Cleaning the fluidics (page 200) for details. At the
end of the cycle, leave the SIP in the tube of water to keep it from
drying out.

Clean the sample stage at shutdown and whenever you see stains
or spills on it.
44 BD Accuri C6 Plus System User’s Guide

Depressing the power button for 3 seconds or longer bypasses the

automatic Clean Fluidics cycle. If you shut down the cytometer in
this way, it does not get properly cleaned, and the software
displays the following message the next time you turn on the
cytometer:

Extra startup time needed due to cleaning or improper shutdown.

When this occurs, the cytometer takes additional time to run the
Clean Fluidics cycle at startup. The startup time can take
approximately 25 minutes.

Shutting down the To shut down the cytometer when running manually:
cytometer when 1. Use a disposable towel or wipe moistened with BD FACSClean
running manually or a 10% bleach solution to wipe down the sample stage.
Follow with a wipe moistened with water.

2. Place a tube containing 2 mL of DI water on the SIP.

a. Push the sample stage back.

b. Place the tube of water over the SIP.
c. While holding the tube, pull the sample stage forward to
support the tube.
3. Press the power button on the front of the cytometer to turn it
off. Leave the tube of water on the SIP.

The Clean Fluidics cycle runs for approximately 13 minutes, then

the cytometer automatically powers off.

Shutting down the To shut down the cytometer when using the CSampler Plus:
cytometer when 1. Ensure the following cleaning tubes are loaded in the
using the CSampler designated fixed locations on the CSampler Plus tray:
Plus
• A tube with 2 mL of BD FACSClean in the triangle ( )
• A tube with 2 mL of DI water in the circle ( )
• A tube with 2 mL of DI water in the square ( )
 Chapter 3: Startup and shutdown 45

2. Press the power button on the front of the cytometer to turn it

off. The SIP is left in the tube of water in the square ( )
location.

The Clean Fluidics cycle runs for approximately 13 minutes, then

the cytometer automatically powers off.

Exiting the To exit the software:

software 1. Select File > Quit.

2. When prompted to save changes to the workspace:

• Click Yes to save changes.

• Click No to close the software without saving changes.
• Click Cancel to cancel the exit and keep the software open.

More information • Cleaning the fluidics (page 200)

46 BD Accuri C6 Plus System User’s Guide
 4
Quality Control
This chapter covers the following topics:

• QC overview (page 48)

• Running instrument QC (page 49)
• Adjusting the bead regions and markers (page 53)
• Viewing QC results (page 58)
• Viewing Levey-Jennings plots (page 63)
48 BD Accuri C6 Plus System User’s Guide

QC overview
Introduction This topic describes the quality control module and recommended
workflow.

About quality The instrument QC module allows you to perform quality control
control on the system. Run quality control daily using the BD CS&T RUO
beads to check and monitor the instrument’s performance. The
CS&T RUO beads have a known median fluorescence intensity
(MFI) and distribution (rCV), and allow you to characterize, track,
and report measurements made by the cytometer.

During instrument QC, the software sets regions around the dim
beads and the mid + bright beads. The locations of the regions are
based on target values, not on the actual locations of the bead
populations. The system measures the brightness and distribution
of the bright beads and compares the results to expected values.
Instrument sensitivity is also calculated. In addition, the
compensation values are updated based on the CS&T RUO bead
results. When the QC test is complete, a Passed or Failed result is
displayed.

Daily workflow The following table shows the recommended daily workflow for
running QC and samples.

Step Description
1 Prepare BD CS&T RUO beads according to the BD CS&T
RUO Beads data sheet.

2 Run BD CS&T RUO beads and optimize the regions and

markers. See Running instrument QC (page 49) and
Adjusting the bead regions and markers (page 53).
 Chapter 4: Quality Control 49

Step Description
3 Prepare samples. See the appropriate reagent kit package
insert or data sheet.

4 Run samples. See Manual data acquisition (page 67) if

running samples manually. See CSampler Plus data
acquisition (page 87) if using a CSampler Plus

5 Analyze data. See Data analysis (page 119).

More information • Running instrument QC (page 49)

• Viewing QC results (page 58)
• Viewing Levey-Jennings plots (page 63)

Running instrument QC
Introduction This topic describes how to run the BD CS&T RUO beads to
check the instrument performance.

Before you begin Prepare BD CS&T RUO beads according to the instructions in the
BD CS&T RUO Beads data sheet.

If you are using the Selectable Lasers Module, the laser will
automatically switch to the standard 3 blue 1 red configuration for
instrument QC. Ensure that the standard optical filters are
installed when running instrument QC. When QC is complete and
you close the QC module, the system will switch back to the
configuration that was previously selected, if different from 3 blue
1 red.
50 BD Accuri C6 Plus System User’s Guide

Procedure To run instrument QC:

1. Click Instrument QC in the upper-right corner of the
workspace.

The QC module opens.

2. Select the bead lot from the BD CS&T Bead Lot menu.

The last bead lot run appears as the default.

If you need to install a bead lot file for a new lot of beads, see
Install a new bead lot (page 51).

3. Enter your operator ID and the lab director ID.

Once the lab director ID is entered, it becomes the default until

you change it.
 Chapter 4: Quality Control 51

4. Run the CS&T RUO beads.

• If you are running bead samples manually, mix the tube of

CS&T RUO beads and load it on the instrument. Click
RUN.
• If you are using a CSampler Plus, click Eject Rack. Mix the
tube of CS&T RUO beads and place it in location A1.
Place the tube rack on the CSampler Plus and click RUN.
5. Wait for acquisition to complete.

The system starts by measuring the background instrument

noise. During this step, the Instrument Noise Measurement
plots are displayed.

After assessing the noise, the system acquires 25,000 events

and displays the BD CS&T Bead plots.

If you want to abort the run, click ABORT QC.

When acquisition is complete, the QC Report table is

displayed, and the result appears at the top of the screen.

6. Rinse the SIP.

If you are running manually, place a tube of DI water on the

SIP. If you are using a CSampler Plus, the instrument
automatically performs a SIP rinse.

7. Proceed to Adjusting the bead regions and markers (page 53).

Install a new bead If this is the first time you are running a bead lot, you will need to
lot install the BD CS&T RUO bead lot file. The bead lot file contains
information specific to the given lot of beads, such as expiration
date, rCV, target MFIs, and sensitivity specifications. Once
installed, the bead lot number will be available to select from the
BD CS&T Bead Lot menu for subsequent QC runs.

To install the bead lot:

1. Select Install from the BD CS&T Bead Lot menu.
52 BD Accuri C6 Plus System User’s Guide

The Install New Bead Lot dialog opens.

2. You can either locate a bead lot file on your computer or scan
the bead lot barcode.

• To browse for a file on your computer, first download the

BD CS&T RUO bead lot file from the BD website. See the
BD CS&T RUO Beads data sheet for instructions on
downloading bead lot files. Once downloaded, select Install
bead lot, then click Choose file. Navigate to the bead lot
file and click Open.
• To scan a bead lot, select Scan bead lot bar code, then scan
the bar code on the box. The bead lot appears in the space
provided.
3. Click Install.

More information • Adjusting the bead regions and markers (page 53)
• Viewing QC results (page 58)
• Removing bead lots from the CS&T bead lot menu (page 185)
• QC troubleshooting (page 231)
 Chapter 4: Quality Control 53

Adjusting the bead regions and markers

Introduction This topic describes how to adjust the regions and markers for the
BD CS&T RUO beads.

The software sets the region and markers for the bright beads
based on target values, not on the actual locations of the
populations. Therefore, you will need to check and optimize the
region and markers, as necessary.

About adjusting Once the bead acquisition is complete and while the QC results are
the regions and displayed, you can adjust the regions and markers to optimize
markers them.

Note: Do not adjust the markers for the noise peaks.

Once you close the QC window or click on the History: Levey-

Jennings tab, you can no longer return to the Instrument QC tab to
edit the regions and markers. A dialog is displayed to remind you.

To adjust the regions and markers, scroll up to the bead plots, as

shown.
54 BD Accuri C6 Plus System User’s Guide

Adjusting the Adjust both the dim and mid + bright regions.
regions in the dot
plot To adjust the bead regions:
1. Use the zoom tool to zoom in on the bead data before
adjusting the regions. See Zooming plots (page 56).

2. Click the bead region to select it.

 Chapter 4: Quality Control 55

The region appears with a bold red outline and eight handles.

Unzoomed Zoomed

3. Adjust the region for the dim beads and the region for the
mid + bright beads. Exclude the doublet population to the
right of the mid + bright beads.

• Place the cursor over a handle. A double-sided arrow

appears. Click and drag to adjust the region in either
direction. When you adjust a corner handle, the region
automatically snaps to a rectangle.
• Place the cursor over an area that is not a handle. A four-
sided arrow appears. Click and drag to move the entire
region in any direction.
If you want to revert to the original regions, click Undo Manual
Adjustments in the upper-right corner of the screen.

Adjusting the To adjust the bright-bead peak markers in all four histograms:
markers in the 1. Use the zoom tool to zoom in on the bead populations before
histogram plots adjusting the markers. See Zooming plots (page 56).

2. Click the bright bead marker to select it.

56 BD Accuri C6 Plus System User’s Guide

The marker appears with a bold red outline and two handles.

Unzoomed Zoomed

3. Adjust the markers.

• Place the cursor over a handle to drag it to the left or right.

• Place the cursor over the horizontal line between the
handles to move the entire marker in any direction.
4. Repeat as necessary to adjust the marker to encompass the
bright bead population, while excluding the doublets.

If you want to revert to the original markers, click Undo Manual

Adjustments in the upper-right corner.

Zooming plots You can zoom in on data to make it easier to see where to set the
markers. If the plots are zoomed when you close the QC report,
the report will be saved with the zoomed plots.

To zoom the plot:

1. Click the zoom tool below the plot to zoom in on the data.

zoom expand
tool tool
 Chapter 4: Quality Control 57

2. Click and drag over the area that you want to zoom in on.
Repeat as necessary.

• To zoom a dot plot, drag in any direction to encompass the

population of interest.
• To zoom a histogram, drag across the peak in either
direction.
Unzoomed Zoomed

3. Click the expand tool to zoom out once. Or, click the Default
Zoom button to go back to the original data range.

Next step • If the result passed, you are ready to run test samples. See
Manual data acquisition (page 67) or CSampler Plus data
acquisition (page 87).
• If the result failed, even after you adjusted the bead regions
and markers, check the QC messages for additional help. See
QC troubleshooting (page 231) for possible causes and
solutions.
• To rerun the beads, mix the tube, click New QC, then click
RUN.
58 BD Accuri C6 Plus System User’s Guide

Viewing QC results
Introduction This topic describes the information that appears on the QC results
screen.

About instrument The instrument QC results are displayed as Passed or Failed. If the
QC results result fails, adjust the mid + bright bead region and the
fluorescence markers. The QC messages that appear below the QC
Report (results) table provide information on the conditions that
the system encountered.

Caution! Do not run process controls or test samples if

instrument QC fails. A successful instrument QC is
required to ensure accurate results.

QC results screen While the QC results window is open, you can make adjustments
to the regions and markers to optimize them. See Adjusting the
bead regions and markers (page 53). Once you close the window
or click on the History: Levey-Jennings tab, the QC results are
locked and no longer editable; however you can add comments.
You can also open and view previous reports and enter comments.
 Chapter 4: Quality Control 59

Instrument noise The system measures the background instrument noise and
measurement plots displays this in the Instrument Noise Measurement plots. Markers
are automatically set in the lower channels around the target
locations for noise peaks. Do not adjust the noise peak markers.

BD CS&T Beads The system acquires 25,000 total events and displays the data in
plots the BD CS&T Beads plots. Regions are set around the dim bead
population and the mid + bright bead population in the dot plot
based on target values. Markers are set around the bright bead
target location in the histogram for each fluorescence parameter.
You can adjust the regions and/or the histogram markers. See
Adjusting the bead regions and markers (page 53) for information.

QC Report table The QC Report table displays the values and Pass/Fail result for
each parameter. If any of the results for an individual parameter
60 BD Accuri C6 Plus System User’s Guide

fails, the failed parameter result appears in red and the overall
instrument QC result fails.

The QC Report table provides values for the following

measurements for FSC, SSC, and FL1–FL4 parameters.

Measurement Description
Bright Bead Median Measured median value for the bright bead
population

MFI Range Target median scatter and fluorescence

intensity range for the bright bead population

% Bright Bead rCV rCV for the bright bead population

Instrument Sensitivity Resolution between instrument noise and

bright bead peak

Sensitivity Spec. Minimum sensitivity required

Parameter Pass/Fail Passed/Failed result for each parameter

QC Messages QC messages, listed below the QC Report table, provide

information about the run. The messages can be helpful in
troubleshooting if instrument QC fails. If there are QC messages
for the run, a QC Messages link appears below the Passed/Failed
results at the top of the screen. Click the link to quickly scroll to
the QC messages. See QC Messages in QC troubleshooting
(page 231) for more information.
 Chapter 4: Quality Control 61

Comments The Comments section allows you to add comments about the run.
You can also add comments before you run the beads or after the
QC window is closed, by viewing previous QC results.

To add comments to the QC report:

1. Click in the Comments text box and type the comment(s).

Viewing previous You can view the QC report for any previous QC run.
QC results
To view QC results from a previous run:
1. Click View Previous QC in the upper-right corner of the
Instrument QC tab.

A window opens showing all QC runs by date.

62 BD Accuri C6 Plus System User’s Guide

2. Select a run, then click OK to view the results for that run.

Note: Results from previous runs are not editable. However,

you can add comments to the report.

For information on deleting old QC runs from the View Previous

QC window, see Removing QC report files from the previous QC
list (page 184).

Printing QC reports You can print QC reports after the run is complete. You can also
print the reports from previous QC runs.

To print the QC report:

1. Click Print in the upper-right corner of the QC window.

Next step • If instrument QC passed, you are ready to run controls and
test samples. See Manual data acquisition (page 67) or
CSampler Plus data acquisition (page 87). Prepare the control
along with the test samples. See the appropriate reagent
package insert or data sheet for use for sample preparation
information.
• If instrument QC still fails after adjusting regions and markers,
check the QC messages at the bottom of the screen for more
information. Refer to QC messages (page 232) for possible
causes and solutions.

More information • Running instrument QC (page 49)

• Adjusting the bead regions and markers (page 53)
• Viewing Levey-Jennings plots (page 63)
• Removing QC report files from the previous QC list
(page 184)
 Chapter 4: Quality Control 63

Viewing Levey-Jennings plots

Introduction This topic describes the History: Levey Jennings tab used to view a
history of QC results and print Levey-Jennings reports.

About Levey- Levey-Jennings reports track QC data over time, allowing you to
Jennings reports view the system’s performance and ensure that the system is
reproducing consistent results. The graphs in the report show you
random errors or shifts and trends in the data for each parameter,
and help you diagnose possible problems with the system.

The graphs show the bright bead median values, %rCV, and
sensitivity with standard deviations for FSC, SSC, FL1, FL2, FL3,
and FL4.

Reviewing the To view the Levey-Jennings report:

Levey-Jennings 1. Click the History: Levey-Jennings tab from the QC window.
report
The Levey-Jennings tab is displayed.
64 BD Accuri C6 Plus System User’s Guide

2. Select the number of days from the View menu in the upper-
left corner of the tab.

You can view data for the last 30, 60, or 90 days. Use the
scroll arrows to the right of the View menu to change the start
date.

• Click the left arrow to select a date 1 month earlier.

• Click the right arrow to select a date 1 month later.

3. Scroll down to view all the graphs for FL1–FL4, FSC, and SSC
median, FL1–FL4 rCVs, and sensitivity for each parameter.
 Chapter 4: Quality Control 65

The graphs show CS&T RUO bead data plotted over time. If
multiple bead lots were used, a different symbol and different
color line appear for each lot.

The horizontal lines above and below the median line

represent ±1 SD and 2 SD. The top and bottom edges of the
graph represent ±3 SD.

A legend in the lower-left corner displays the symbols used for

invalid results, where a data point was collected but the value
was not used in the SD calculation. The different symbol
shapes correspond to the symbols used for different bead lots.

4. To close the QC window, click Close in the lower-right corner.

Printing Levey- To print a Levey-Jennings report:

Jennings reports 1. Select the number of days you want to plot from the View
menu.

2. Click Print in the upper-right corner of the Levey-Jennings tab.

More information • Viewing QC results (page 58)

• QC troubleshooting (page 231)
66 BD Accuri C6 Plus System User’s Guide
 5
Manual data acquisition
This chapter covers the following topics:

• Collect tab (page 68)

• Setting acquisition parameters (page 72)
• Run settings (page 73)
• Fluidics rate (page 75)
• Threshold (page 79)
• Running samples using the Collect tab (page 81)
• Ending a data collection session (page 84)
68 BD Accuri C6 Plus System User’s Guide

Collect tab
Introduction This topic describes the Collect tab in BD Accuri C6 Plus software.

About the Collect The Collect tab allows you to set data collection criteria, start and
tab stop data acquisition, and view data on collected samples. You can
use pre-defined templates for acquisition or create your own
workspace with custom acquisition criteria. The tab contains
buttons and controls for performing the following functions:

• Acquiring samples
• Creating plots (histogram, dot, or density) for viewing data
• Setting stop criteria (run limits) and thresholds
• Controlling the fluidics
• Using regions and markers to create gates and obtain statistics
• Saving and print plots and data
• Accessing the following analytic functions:
– Creating nested gates
– Setting and/or adjusting fluorescence compensation
– Generating statistics
 Chapter 5: Manual data acquisition 69

Viewing the Collect The Collect tab is displayed when the software opens. You can also
tab view the tab by clicking on Collect from any of the other tabs
(Analyze, Statistics, or Batch Analysis).

The Collect tab is organized into two major sections:

• Instrument Control Panel. Panel on the left side of the window

that contains controls for collecting data.

• Data display. Area on the right that shows sample data in plots
and in a statistics table.
70 BD Accuri C6 Plus System User’s Guide

Collect tab controls The following table describes each of the controls and indicators in
the Collect tab:

Control Description
Sample naming field Text box for naming the sample.

Sample grid Matrix laid out in the configuration of a 96-well plate to help organize
experiments and collect data from sample tubes. Each sample is
acquired into its own well in the sample grid. The color-coded wells can
be filled with data in any order.
White. Does not contain data.
Blue. Contains data.
Red outline. Currently selected for viewing or collecting data.

Traffic light and Indicator that displays software readiness and system messages. Before
message data collection can begin, the software must display a green traffic light
with the message Cytometer is connected and ready.
The traffic light status is color-coded:
Green. Software is ready to collect data or is collecting data.
Yellow. Cytometer is preparing to perform an action or a non-critical
error has occurred.
Red. Critical error has occurred.

Run Settings Contains a set of controls that allow you to define criteria for
automatically stopping data collection. See Run settings (page 73) for
more information.

Backflush Performs a backflush to force sample out of the SIP.

SIP Clean Performs a SIP Clean to clean the inside of the SIP.

Fluidics Contains a set of controls for defining flow rate and core size. See
Fluidics rate (page 75) for more information.

Threshold Allows you to set the event threshold to eliminate debris and noise from
sample data. The default value is 80,000 on FSC-H. See Threshold
(page 79) for more information.
 Chapter 5: Manual data acquisition 71

Control Description
RUN/PAUSE/ADD Button that performs the following functions:
TO
RUN. Starts sample acquisition.
PAUSE. Pauses acquisition. Click ADD TO to resume acquisition.
ADD TO. Allows you to collect more sample data in a well that already
contains data.

Set Color Opens the Color Compensation dialog for correcting fluorescence
Compensation spillover. See Correcting fluorescence spillover (page 149) for more
information.

Acquisition Displays the following information about the most recent acquisition
counters for the selected well (Last Run) and all acquisitions for the selected well
(Cumulative) in real-time:
Events. Number of events acquired.
Time. Elapsed acquisition time.
Microliters. Volume of sample acquired.
Events/Sec. Events acquired per second. When the run is completed,
this is the average value.
Events/µL. Events acquired per microliter. When the run is completed,
this is the average value.

Delete Events Permanently deletes all events from the current sample. Select the show
warning checkbox to display a warning message before deleting sample
data. Also contains a Data Capacity Used meter that displays the
amount of data storage capacity currently used in the software.

Plots pane Area displaying two rows of plot corrals for graphically viewing data
for the selected sample. Scroll up or down to view additional plots. You
can create multiple plots for each sample. Each plot corral contains
buttons for creating histogram, dot, and density plots. See Plots and
gates (page 156) for more information on plots.

Statistics table Table below the plots that displays statistical information on all plots.
72 BD Accuri C6 Plus System User’s Guide

Data collection Complete the following workflow steps to collect sample data.
workflow
Step Description
1 (Optional) Open a template. See Creating a workspace
template (page 189).

2 Set the acquisition parameters—run limits, fluidics rate, and

threshold. See Setting acquisition parameters (page 72).

3 Open plots, create and apply gates. See Creating a new plot
(page 157) and Creating and applying gates (page 160).

4 Run the sample. See Running samples using the Collect tab
(page 81).

5 (If needed) Apply fluorescence compensation values. See

Correcting fluorescence spillover (page 149).

6 Save the data. See Workspace files (page 186).

Setting acquisition parameters

Introduction This topic lists the different acquisition parameters. See the
following sections for information on each parameter.

Acquisition You can set the following acquisition parameters:

parameters
• Run settings (page 73)
• Fluidics rate (page 75)
• Threshold (page 79)
 Chapter 5: Manual data acquisition 73

Run settings
Introduction This topic explains run settings and provides instructions for
enabling and disabling run limits for data collection.

About run settings Set a run limit to indicate when to stop collecting data. You can set
a run limit based on any of the following criteria:

• After a specified number of events

• After a specified time
• After a specified volume
• For an unlimited time (until you manually stop the run)
You can choose multiple run limits (collection stops when the first
limit is reached). You can also change the run limit once a gate has
been set on the population of interest.

Disabling run limits To collect samples without setting a run limit:

1. Select the Run Unlimited checkbox.
74 BD Accuri C6 Plus System User’s Guide

Setting an event To stop the run after a specified number of events have been
run limit collected:
1. Select Run with Limits.

2. Select the checkbox next to the events field.

3. In the associated text box, type the number of events at which

to stop the run.

4. Do one of the following in the list below the text box:

• Select Ungated Sample.

• Select a gating strategy (if one exists) to stop the run when
the assigned number of events have been collected in the
gating region.
5. To set a live gate, select the Do not collect events outside
checkbox. Then, select the gate from the menu. See Creating a
live gate (page 166).

This option is called Only collect events inside in the Auto

Collect tab when using a CSampler Plus.

Setting a time run To stop the run after a time has expired:
limit 1. Select Run with Limits.

2. Select the checkbox next to the Min and Sec fields.

3. Type the number of minutes (min) and seconds (sec) at which

to stop the run.

Setting a volume To stop the run after a specified volume has been collected:
run limit 1. Select Run with Limits.

2. Select the checkbox next to the µL field.

3. Type the volume in microliters (µL) at which to stop the run.

 Chapter 5: Manual data acquisition 75

Fluidics rate
Introduction This topic describes how to set the fluidics rate for data collection.

About fluidics rates The system can accommodate an upper limit of 10,000 events per
second, but we recommend acquiring samples at a rate of 2,500
events per second or less to ensure the best data resolution.

Three preset rates are provided—Slow, Medium, and Fast. You can
also customize the fluidics rate by selecting a rate outside the preset
values.

Setting the fluidics To set the fluidics rate:

rate 1. Select Slow, Medium, or Fast under Fluidics in the Instrument
Control Panel.

• Slow is 14 µL/min.
• Medium is 35 µL/min.
• Fast is 66 µL/min.
Note: We recommend starting data collection on Slow and
observing the event rate. You can then switch the setting to
Medium or Fast, if necessary.

2. Observe the event rate. If the rate is near or greater than

10,000 total events per second on the Slow setting, consider
the following options:

• Increase the primary threshold value, taking care that the

increase does not remove cells of interest from the data set.
• Include a secondary threshold, taking care not to exclude
cells of interest.
• Dilute the sample.
76 BD Accuri C6 Plus System User’s Guide

Customizing the Advanced users can set a custom fluidics rate.

fluidics rate
To customize the fluidics rate:
1. Select Custom under Fluidics in the Instrument Control Panel.

2. Move the Custom slider to adjust the flow rate.

Adjusting the core Advanced users can adjust the core size of the sample stream for a
size of the sample range of cell sizes.
stream
To customize the sample core size:
1. Click Set Core Size under Fluidics in the Instrument Control
Panel.

2. Move the slider to adjust the core size.

 Chapter 5: Manual data acquisition 77

3. Click OK to set the core size and close the dialog.

Note: Certain core sizes are not compatible with certain flow
rates, and the software does not allow these combinations to
be set. Use the following table to determine allowable
combinations.

Minimum Maximum
Core size flow rate flow rate
5 10 11

6 10 16

7 10 22

8 10 29

9 10 36

10 10 45

11 10 54

12 10 65

13 10 76

14 12 88

15 14 100

16 15 100

17 17 100

18 19 100

19 22 100

20 24 100

21 26 100

22 29 100

23 32 100

24 35 100
78 BD Accuri C6 Plus System User’s Guide

Minimum Maximum
Core size flow rate flow rate
25 38 100

26 41 100

27 44 100

28 47 100

29 50 100

30 54 100

31 58 100

32 61 100

33 65 100

34 69 100

35 74 100

36 78 100

37 82 100

38 87 100

39 91 100

40 96 100
 Chapter 5: Manual data acquisition 79

Threshold
Introduction This topic explains the threshold and provides instructions for
setting a collection threshold.

About thresholds Thresholds eliminate debris and noise from cell samples so that
sample data are not compromised by non-cellular events. By
default, events lower than channel 80,000, for the selected
parameter, are filtered out.

You can change the threshold settings at any time before, during,
or after data acquisition. The primary threshold is the parameter
that triggers data collection. FSC-H is the default primary
threshold. You can optionally set a secondary threshold to filter
out additional data.

The most consistent, predictable results will be obtained if

threshold settings are chosen before final data collection for any
given experiment.

All thresholds are set on the Height signal for any given parameter.
For best results when setting or changing thresholds, create a plot
that displays the Height signal for the threshold channel and
observe the effect on the data as the threshold is raised or lowered.

Caution Take care when setting the thresholds before or

during data collection. Any event not meeting the
threshold criteria will be not be acquired or saved. When
changes are made to the threshold values after data
collection, the software displays a warning message if the
new threshold value will result in permanent data loss.
80 BD Accuri C6 Plus System User’s Guide

Setting the To set the threshold:

threshold 1. Click Set Threshold in the Instrument Control Panel.

2. Select the primary threshold parameter from the Primary

Threshold list.

3. Type 80000 in the less than text box to set the threshold
minimum to channel 80,000.

You may need to set a lower or higher FSC-H threshold when

working with small cells (such as platelets or bacteria) or large
cells (such as cell lines), respectively.

4. If you want to apply a secondary threshold for filtering out

more data, do the following:

• Select the threshold parameter from the Optional

Secondary Threshold list.
• Type a value in the less than text box to set the threshold
minimum.
5. Select one of the following Apply to:

• Only this sample to apply settings to the current sample

only.
• All samples to apply the settings to all samples, including
all previously collected data in other data wells.
6. Click Apply to apply the threshold settings.

7. Click Close to close the dialog.

 Chapter 5: Manual data acquisition 81

Running samples using the Collect tab

Introduction This topic describes how to run a sample after all acquisition
parameters (run settings, fluidics rate, and threshold) are set.

About collecting • Before data collection can begin, the software must display a
sample data green traffic light with the message Cytometer is connected
and ready.
• Each data well holds a maximum of 1 million events. You can
add more events (up to a total of 1 million) to a well already
containing data.
• When a well already contains data, the RUN button becomes
ADD TO.
Note: Run limits may need to be adjusted to accommodate
additional data.

Before you begin Set the acquisition parameters. See Setting acquisition parameters
(page 72).

Procedure To run a sample:

1. Select an empty sample well.

A1 is selected by default.

2. Enter a sample name in the text box above the 96-well grid.
82 BD Accuri C6 Plus System User’s Guide

You can name samples at any time. If you do not type in a

name, the sample is named according to the well location (for
example, A01).

3. Resuspend the cells in the sample tube and place the tube on
the SIP.

4. Click RUN to start the sample collection.

If you have not yet saved the workspace, you will be prompted
to do so.

Fluidics initialization begins. During this time the traffic light

turns yellow and the message Preparing to analyze sample is
displayed.

Once initialization is complete, the traffic light turns green and

the message Cytometer is running is displayed.

The well flashes blue during data collection. After the run limit
is reached, the well stops flashing and remains blue, indicating
that the well contains data.

5. If you are running unlimited, click PAUSE to stop acquisition.

Note: If desired, you can collect more data in a well that

contains data at any time by selecting the well and clicking
ADD TO. See Adding more sample data to a file (page 83).

6. To run the next sample, click the next well in the sample grid.
If necessary, adjust the acquisition parameters, then click
RUN.

To have the next sample well automatically selected, select

Display > Auto-Select Next Well. Choose horizontally or
vertically and click OK. When the run limit for a sample is
reached, the next sample well is automatically selected. You
 Chapter 5: Manual data acquisition 83

need only click RUN. If you are running unlimited, the

collection will stop when 1 million events are acquired.

Adding more You can collect new samples and add the data to a workspace file
sample data to a that already contains sample data, by adding data to a well that
file already contains data or to an empty well. If the well already
contains data, you may need to increase the number of events
defined for the run limit to allow for more events to be acquired.

To add data to a file:

1. Resuspend the sample and place the tube on the SIP.

2. Click a data well in the 96-well sample grid.

If you select an empty well, any plots and gates you created
earlier are still displayed, but they do not contain data.

3. Click RUN (or ADD TO) to start acquisition.

The cytometer stops sampling from the tube when the run
limit is reached.

Caution If you click ADD TO, the software will

collect data into a well that already contains data.
84 BD Accuri C6 Plus System User’s Guide

Pausing data You can interrupt sample acquisition any time during a run.
collection
To stop a run:
1. Click PAUSE.

To restart the run:

1. Click ADD TO. The software resumes data collection in the
current well.

If you want to delete the data that was already collected before
you paused, click Delete Events at the bottom of the
Instrument Control Panel, then click RUN to acquire new
data.

Next step Ending a data collection session (page 84)

More information • Creating a live gate (page 166)

Ending a data collection session

Introduction This topic describes how to save the workspace file and clean the
SIP at the completion of a run.

Auto-save data If you have the Auto-save Data Settings feature enabled, the
feature software will automatically save the workspace file at the end of
the run. If you make any changes to the workspace after the run is
finished, the changes will not be automatically saved. You will
need to manually save the file. See Saving the workspace file
(page 85).

To enable the auto-save data feature:

1. Select File > Auto-save Data Settings.
 Chapter 5: Manual data acquisition 85

2. Select the enabled option and click OK.

Saving the To save the workspace file at the end of the run:
workspace file 1. Select File > Save.

Performing a SIP When you finish collecting samples, clean the SIP to ensure cells or
clean other particles are not left in the SIP.

To clean the SIP:

1. Click SIP Clean in the instrument control panel.

A dialog prompts you to load a tube containing 2 mL of

BD™ FACSClean.

2. Load the tube of BD FACSClean and click SIP Clean.

When step 1 is complete, a dialog prompts you to load a tube

containing 2 mL of water.

3. Load the tube of water and click SIP Clean.

86 BD Accuri C6 Plus System User’s Guide
 6
CSampler Plus data acquisition
This chapter covers the following topics:

• Manual Collect tab (page 88)

• Running samples using the Manual Collect tab (page 93)
• Auto Collect tab (page 99)
• Creating sample sets for Auto Collect experiments (page 103)
• Setting parameters for Auto Collect experiments (page 106)
• Running samples using the Auto Collect tab (page 112)
• Ending a data acquisition session (page 117)
88 BD Accuri C6 Plus System User’s Guide

Manual Collect tab

Introduction This topic describes the Manual Collect tab in BD CSampler Plus
software.

About the Manual The Manual Collect tab allows you to set the data collection
Collect tab criteria, start and stop data acquisition, and view data on collected
samples. You can use pre-defined templates for acquisition or
create your own workspace with custom acquisition criteria. The
tab contains buttons and controls for performing the following
functions:

• Run individual samples from a plate or tube rack. Settings

such as thresholds and color compensation can be applied to
either the current sample being viewed or to all the samples in
the sample grid.
• Set up experiments to be collected in the Auto Collect tab. The
following must be set up in the Manual Collect tab before
switching to the Auto Collect tab:
– plots (histogram, dot, or density) for viewing data
– gating strategies
– threshold values
Note: Run Limit settings for auto collect runs must be set in
the Auto Collect tab.

• Collect data from a plate if the plate has been interrupted or

aborted in the Auto Collect tab.
• View sample data in plots and in the Statistics table.
• Print plots and statistics.
• Perform color compensation.
• Import and export data.
 Chapter 6: CSampler Plus data acquisition 89

Viewing the The Manual Collect tab is organized into two major sections:
Manual Collect tab
• Instrument Control Panel. Panel on the left side of the window
that contains controls for collecting data.

• Data display. Large area on the right side of the window that
shows the sample data in plots and in a Statistics table.
90 BD Accuri C6 Plus System User’s Guide

Manual Collect tab The following table describes each of the controls and indicators in
controls the Manual Collect tab.

Control Description
Plate Type List for selecting the plate type.

Load Plate/Eject Moves the sample plate into position to be loaded onto or ejected from
Plate the flow cytometer.

Plate Name Text box for naming the plate.

Sample naming field Text box for naming the sample.

Sample grid Matrix laid out in the configuration of a 96-well plate or a 24-tube
rack to correspond to the sample vessel. Each sample is acquired into
its own well in the sample grid. The color-coded wells can be filled with
data in any order.
White. Does not contain data.
Blue. Contains data.
Red outline. Currently selected for viewing or collecting data.

Traffic light and Indicator that displays software readiness and system messages. Before
message data collection can begin, the software must display a green traffic light
with the message Cytometer is connected and ready.
The traffic light status is color-coded:
Green. Software is ready to collect data or is collecting data.
Yellow. Cytometer is preparing to perform an action, performing an
action other than data collection (such as clean or agitate), or a non-
critical error has occurred.
Red. Critical error has occurred or the CSampler Plus has collided.

Run Settings Contains a set of controls that allow you to define criteria for
automatically stopping data collection. See Run settings (page 73) for
more information.
 Chapter 6: CSampler Plus data acquisition 91

Control Description
SIP Rinse Rinses the outside of the SIP between samples.
Agitate Agitates the plate/rack to keep cells in suspension.
Backflush Forces sample out of the SIP.
SIP Clean Performs a SIP Clean to clean the inside of the SIP.

Fluidics Contains a set of controls for defining flow rate and core size. See
Fluidics rate (page 75).

Threshold Allows you to set the event threshold to eliminate debris and noise
from sample data. The default value is 80,000 on FSC-H. See Setting
the threshold (page 107).

RUN/PAUSE/ADD Button that performs the following functions:

TO
RUN. Starts sample acquisition.
PAUSE. Pauses acquisition. Click ADD TO to resume data collection.
ADD TO. Allows you to collect more sample in a well that already
contains data.

Set Color Opens the Color Compensation dialog for correcting fluorescence
Compensation spillover. See Correcting fluorescence spillover (page 149).

Acquisition counters Displays the following information about the most recent acquisition
for the selected well (Last Run) and all acquisitions for the selected well
(Cumulative) in real-time:
Events. Number of events acquired.
Time. Elapsed acquisition time.
Microliters. Volume of acquired sample.
Events/sec. Events acquired per second. When the run is completed,
this is the average value.
Events/µL. Events acquired per microliter. When the run is completed,
this is the average value.
92 BD Accuri C6 Plus System User’s Guide

Control Description
Delete Events Permanently deletes all events from the current sample. Select the show
warning checkbox to display a warning message before deleting sample
data. Also contains a Data Capacity Used meter that displays the
amount of data storage capacity currently used in software.

Plots pane Area displaying two rows of plot corrals for graphically viewing data
for the selected sample. Scroll up or down to view additional plots. You
can create multiple plots for each sample. Each plot corral contains
buttons for creating histogram, dot, and density plots. See Plots and
gates (page 156).

Statistics table Table below the plots that displays statistical information on all plots.

Manual data Complete the following workflow stages to collect sample data
collection with the BD CSampler Plus software.
workflow
Stage Description
1 In the Manual Collect tab, do one of the following:
 Select a plate type. Name the plate (optional).
 Open a template. See Creating a workspace template
(page 189).

2 Set the acquisition parameters—fluidics rate, threshold, and

run limits.
See Setting acquisition parameters (page 72) for more
information.

3 Open plots, create and apply gates. See Creating a new plot
(page 157) and Creating and applying gates (page 160).

4 Run the sample. See Running samples using the Manual

Collect tab (page 93).

5 (If needed) Apply fluorescence compensation values. See

Correcting fluorescence spillover (page 149).

6 Save the data. See Workspace files (page 186).

 Chapter 6: CSampler Plus data acquisition 93

Running samples using the Manual Collect tab

Introduction This topic describes how to run samples using the Manual Collect
tab after all acquisition parameters are set.

Before you begin Set the acquisition parameters. See Setting acquisition parameters
(page 72).

About collecting • Before data collection can begin, the software must display a
sample data green traffic light with the message Cytometer is connected
and ready.
• Each data well holds a maximum of 1 million events. You can
add more events (up to a total of 1 million) to a well already
containing data.
• When a well already contains data, the RUN button displays
ADD TO.
Note: Run limits may need to be adjusted to accommodate
additional data.

Procedure To manually run the sample with the BD CSampler Plus software:
1. Select a plate type from the Plate Type list.
94 BD Accuri C6 Plus System User’s Guide

The software prompts you to save the workspace file.

The sample grid displays the available wells for data

collection, based on the assigned plate type:

– 96-well plate. Displays wells for rows A-H, columns 1-12

– 48-well plate. Displays wells for rows A-F, columns 1-8

– 24-tube rack. Displays wells for rows A-D, columns 1-6

Note: Changing the plate type automatically opens a new

blank workspace.

2. (Optional) Name the plate by typing a label in the Plate Name

field.

3. Type the sample name into the text box above the 96-well grid.
 Chapter 6: CSampler Plus data acquisition 95

You can name samples at any time. If you do not type in a

name, the sample is named according to the well location (for
example, A01).

4. Resuspend the cells in the sample tube or plate, and load the
tube rack or plate.

5. Select a well in the sample grid that corresponds to the sample

location in the plate or tube rack.

6. Click RUN to start the sample collection.

Fluidics initialization begins. During this time the traffic light

turns yellow and the message Preparing to analyze sample is
displayed.

Once initialization is complete, the traffic light turns green and

the message Cytometer is running is displayed.

The well flashes blue during data collection. After the run limit
is reached, the well stops flashing and remains blue, indicating
that the well contains data.

7. If you are running unlimited, click PAUSE to stop acquisition.

Note: If desired, you can collect more data in a well that

contains data at any time by selecting the well and clicking
ADD TO. See Adding more sample data to a CSampler Plus
file (page 96).

8. To run the next sample, click the next well in the sample grid.
If necessary, adjust the acquisition parameters, then click
RUN.

To have the next sample well automatically selected, select

Display > Auto-Select Next Well. Choose horizontally or
vertically and click OK. When the run limit for a sample is
reached, the next sample well is automatically selected. You
96 BD Accuri C6 Plus System User’s Guide

need only click RUN. If you are running unlimited, the

collection will stop when 1 million events are acquired.

Agitating samples Note: The agitate feature is designed to maintain a sample in

suspension, not to resuspend a completely settled sample.

Caution Sample volume should not exceed 50% well

capacity to effectively maintain suspension while
avoiding spillover during agitation.
To perform an agitate cycle:
1. Click Agitate.

Rinsing the SIP A rinse cycle can be used to rinse the SIP between samples. The SIP
between samples goes to the water tube in the square location of the CSampler Plus
tray and aspirates the contents of the SIP up then out of the SIP,
then rinses the SIP with clean fluid.

To rinse the SIP:

1. Click SIP Rinse.

Adding more You can collect new samples and add the data to a CSampler Plus
sample data to a workspace file that already contains data, by adding data to a well
CSampler Plus file that already contains data or to an empty well.

To add data to a CSampler Plus file:

1. Resuspend the sample and load the plate or tube rack.
 Chapter 6: CSampler Plus data acquisition 97

2. Click a data well in the sample grid.

If you select an empty well, any plots and gates you created
earlier are still displayed, but they do not contain data.

3. Click RUN (or ADD TO) to start acquisition.

The flow cytometer stops sampling from the tube or well when
the run limit is reached.

Caution If you click ADD TO, the software will

collect data into a well that already contains data.

Pausing data You can interrupt sample acquisition any time during a run.
acquisition
To pause a run:
1. Click PAUSE.

To restart the run:

1. Click ADD TO. The software resumes data collection in the
current well.

If you want to delete the data that was already collected before
you paused, click Delete Events, then click RUN to acquire
new data.

Ending a data When you finish collecting samples, clean the SIP to ensure cells or
acquisition session other particles are not left in the SIP.

To clean the SIP:

1. Click SIP Clean.
98 BD Accuri C6 Plus System User’s Guide

A dialog opens prompting you to load tubes of BD FACSClean

and water.

2. Ensure the following tubes are in the designated locations on

the CSampler Plus. If necessary, click Eject Rack/Plate and
insert the tubes, then click Load Rack/Plate.

• 2 mL of BD FACSClean in the triangle location.

• 2 mL of DI water in the circle location.
3. Click SIP Clean.

More information • Creating a live gate (page 166)

 Chapter 6: CSampler Plus data acquisition 99

Auto Collect tab

Introduction This topic describes the Auto Collect tab in the BD CSampler Plus
software.

About the Auto The Auto Collect tab allows automated sampling from a well plate
Collect tab or sample tube rack. The tab contains buttons and controls for
performing the following functions:

• Loading and ejecting a plate

• Creating sets of samples with the same acquisition settings
• Acquiring data
• Setting thresholds and stop criteria
• Controlling the fluidics
• Rinsing the SIP between samples
• Agitating (resuspending) samples
• Viewing sample information

Viewing the Auto The Auto Collect tab is organized into two major sections:
Collect tab
• Instrument Control Panel. Panel on the left side of the window
that contains controls for collecting data.

• Sample Annotation table. Large table on the right side of the

window used to name samples, rename parameters, and add a
notation to each sample.
100 BD Accuri C6 Plus System User’s Guide

Auto Collect tab The following table describes each of the controls and indicators in
controls the Auto Collect tab.

Control Description
Plate Type List for selecting the plate type.

Load Plate/Eject Moves the sample plate into position to be loaded onto or ejected from
Plate the flow cytometer.

Plate Name Text box for naming the plate.

 Chapter 6: CSampler Plus data acquisition 101

Control Description
Sample grid Matrix laid out in the configuration of a multi-well plate to help
organize experiments and collect data from sample tubes or wells. Each
sample is acquired into its own well in the sample grid. The wells are
color-coded:
White. Does not contain data.
Colored fill. Acquisition settings are applied. Each unique acquisition
setting is indicated by a color change.
Small blue box. Contains data.
Red outline. Currently selected for viewing or collecting data.

Traffic light and Indicator that displays software readiness and system messages. Before
message data collection can begin, the software must display a green traffic light
with the message Cytometer is connected and ready.
The Traffic Light status is color-coded:
Green. Software is ready to collect data or is collecting data.
Yellow. Cytometer is preparing to perform an action, performing an
action other than data collection (such as cleaning or agitating), or a
non-critical error has occurred.
Red. Critical error has occurred or the CSampler Plus has collided.

Run Limits Contains a set of controls that allow you to define criteria for
automatically stopping data collection.

SIP Rinse Settings Allows you to select a SIP Rinse cycle to perform between each sample.
See Rinsing the SIP between samples (page 96).

Fluidics Allows you to set the flow rate. See Setting the fluidics rate (page 107).
Custom flow rates are not available in the Auto Collect tab.

Set Threshold Sets the event threshold to eliminate debris and noise from samples.
The default value is 80,000 on FSC-H. You can also set a secondary
threshold. See Threshold (page 79).

Apply Settings/ To apply or remove sample settings, such as run limits, fluidics,
Remove Settings threshold, and SIP rinse settings.

Agitate Plate Performs an agitate cycle to keep particles in suspension. See Agitating
samples (page 108).
102 BD Accuri C6 Plus System User’s Guide

Control Description
SIP Clean Performs a SIP Clean at the end of the run.

Run Horizontally/ Allows you to select how you want to acquire samples—horizontally or
Run Vertically vertically. See Setting the run direction (page 109).

Open Run Display Opens the run display for starting and stopping a run, and viewing
data acquisition counters and two plots of data.

Sample Annotation Contains fields for naming samples, renaming parameters, and adding
table a notation to each sample. Information can be entered manually for
each sample or copied and pasted from a spreadsheet program. See
Creating the Sample Annotation table (page 110).

Auto Collect Use the following workflow to run samples using Auto Collect.
workflow
Stage Description
1 Creating plot displays and gates in the Manual Collect tab.

2 Display the Auto Collect tab and select the plate type.

3 Create sample sets with specified acquisition, agitation,

wash settings, and run direction. See Creating sample sets
for Auto Collect experiments (page 103).
Agitate settings are applied to the entire plate, while
acquisition parameters, wash settings and run limits can be
applied to individual sample sets.

4 Create the Sample Annotation table. See Creating the

Sample Annotation table (page 110).

5 Open the Run Display.

6 Select the plots to view during acquisition. Plots created in

the Manual Collect tab are available for viewing. You can
view two plots at a time in the Auto Collect Run Display.

7 Run the samples. See Running samples using the Auto

Collect tab (page 112)
 Chapter 6: CSampler Plus data acquisition 103

Creating sample sets for Auto Collect experiments

Introduction This topic describes how to create sample sets to use with Auto
Collect.

About sample sets A sample set is a group of samples that use the same data
acquisition criteria (such as number of events or threshold setting).
Acquisition criteria include:

• Run limits (number of total or gated events, sample volume, or

time)
• Fluidics settings
• Wash settings
• Threshold(s) (default is FSC-H 80,000)
Each sample set is highlighted in a different color. Twelve colors
are available, but up to 96 sets can be created (colors are reused).

If you are not creating sample sets and all samples will have the
same collection criteria, proceed to Setting parameters for Auto
Collect experiments (page 106).

Caution When a plate run is started, data will be

collected in every well with settings applied, regardless
of whether the well contains previously acquired data.

Procedure To create sample sets:

1. Select one or more wells to include in the set by doing one of
the following:

• Click on individual wells.

• Click a column (1-12) or row header labels (A-H) to select
the entire column or row. Multiple rows and columns can
be selected simultaneously.
• Click Select All above the sample grid to select all the wells.
104 BD Accuri C6 Plus System User’s Guide

Selected wells contain a black check mark.

2. Enter settings for the following:

• Run limits
• (Optional) SIP Rinse Settings
• Fluidics
• Threshold – default is FSC-H 80,000
See Setting parameters for Auto Collect experiments
(page 106) for more information.

If run limit or fluidics settings were modified in the Manual

Collect tab, those settings do not carry over in the Auto
Collect tab. Threshold settings and color compensation
settings do carry over.

3. Click Apply Settings.

The selected samples are highlighted in one color. If you have

not already saved the workspace file, you will be prompted to
name the file and designate a location.
 Chapter 6: CSampler Plus data acquisition 105

All sample settings are saved each time you click Apply
Settings. Acquisition settings displayed in the Control Panel do
not change. To see the actual settings for a specific set, press
Ctrl and click a well of interest within the set.

4. If applicable, select the next set of samples, set the acquisition

parameters, and click Apply Settings.

The selected samples are highlighted in a new color.

Viewing sample To view data acquisition settings for a particular well:

settings 1. Press Ctrl and click a well of interest.

Modifying sample To modify a set after it is created:

settings 1. Do one of the following:

• Double-click on a well in the set to select the entire set.

• Select one or more wells within the set.
2. Adjust the data acquisition settings.
106 BD Accuri C6 Plus System User’s Guide

3. Click Apply Settings. The selected wells change to a new color.

Removing sample To remove settings from a sample set:

settings 1. Do one of the following:

• Double-click on a well in the set to select the entire set.

• Select one or more wells in the set.
2. Click Remove Settings.

Each selected sample well reverts to a white box and is

removed from the set.

Acquisition settings displayed in the Control Panel do not

change.

Setting parameters for Auto Collect experiments

Introduction This topic describes how to set the run limits, fluidics rate,
threshold, agitate, and run direction settings for Auto Collect
experiments.

Setting run limits The run limit defines when data acquisition will stop. The
following parameters can be used individually or in combination to
set a run limit.

• Number of events (total or in a specified gate)

• Time
• Volume
If multiple run limits are set, data collection stops on
whichever limit is reached first. If run limit settings were
modified in the Manual Collect tab, they do not carry over in
the Auto Collect tab.

See Run settings (page 73) for more information about setting
run limits.
 Chapter 6: CSampler Plus data acquisition 107

Setting the fluidics The cytometer can accommodate an upper limit of 10,000 events
rate per second, but it is recommended to acquire samples at a rate of
2,500 events per second or less to ensure the best data resolution.

If fluidics settings were modified in the Manual Collect tab, they

do not carry over in the Auto Collect tab.

To set the fluidics rate:

1. Click the Slow, Medium, or Fast button in the Fluidics section
of the Auto Collect tab.

• Slow is 14 µL/min.
• Medium is 35 µL/min.
• Fast is 66 µL/min.
You cannot customize the flow rate or the sample stream core
size in Auto Collect experiments.

Setting the Use thresholds to eliminate light scatter and/or fluorescence signals
threshold caused by debris in cell samples and electronic noise inherent in the
system.

To set the threshold:

1. Select the threshold parameter and enter the channel value in
the less than text box.

2. (Optional) Select a secondary threshold parameter and enter

the channel value in the text box.

Both thresholds are applied to every event.

See Threshold (page 79) for more information about setting

thresholds.

Rinsing the SIP Multiple rinse cycles of the SIP can be specified in the Auto Collect
between samples tab. Up to three rinse cycles can be specified at a time.

• One wash cycle typically reduces carryover to < 1.0%.

• Two wash cycles typically reduce carryover to < 0.1%.
108 BD Accuri C6 Plus System User’s Guide

The specified SIP rinse cycles are performed after each sample
acquisition, and can be applied to individual wells.

To rinse the SIP:

1. Select the number of cycles from the SIP Rinse Settings menu.

The number of SIP rinse cycles you select will be performed

after every sample.

Agitating samples The agitate feature is designed to keep samples in uniform

suspension during data collection. Agitate gently shakes the sample
plate/tube rack in 15-second cycles.

One, two, or three agitation cycles can be automatically performed

from the Auto Collect tab during the plate run.

You can specify whether to agitate after a given number of samples

or in intervals from 1 to 30 minutes. If the interval is reached
during acquisition, the cytometer will complete data acquisition
before performing the agitation.

Agitation is only performed when the SIP is clear of any well or

tube and does not interrupt data collection.

Caution Sample volume should not exceed 50% well

capacity to effectively maintain suspension and avoid
spillover during agitation. For best results, use 96-well
U-bottom plates or standard 12x75-mm tubes.
Agitate settings are applied to the entire plate or tube rack and
cannot be applied to individual wells.

To set an agitate cycle:

1. Select one of the following options:
 Chapter 6: CSampler Plus data acquisition 109

• To agitate before a given number of wells, click the top

button.
• To agitate after a given number of minutes, click the
bottom button.
2. Select the number of 15-second agitate cycles (1, 2, or 3).

3. Do one of the following:

• Type in the number of wells to increment between agitate

cycles.
• Type in the number of minutes to increment between
agitate cycles.

Setting the run Data can be collected from plates either horizontally (A1, A2, A3,
direction etc) or vertically (A1, B1, C1, etc).

To set the run direction:

1. Do one of the following:

• Click Run Horizontally to collect the samples horizontally.

• Click Run Vertically to collect the samples vertically.
110 BD Accuri C6 Plus System User’s Guide

Creating the Use the Sample Annotation table to name samples, rename
Sample Annotation parameters, and add a notation to each sample. Information can be
table entered manually for each sample or copied and pasted from a
spreadsheet program.

To manually create the Sample Annotation table:

1. For each sample well, click in each cell and type the
appropriate information. The parameter names entered in the
Sample Annotation table will also be applied to data plots.

To copy and paste information from a spreadsheet:

1. Highlight fields in the spreadsheet.

2. Press Ctrl + C to copy.

3. In the Sample Annotation table, click on Sample Name of well

A1 (or other appropriate cell).

4. Press Ctrl + V to populate the Sample Annotation table with

the highlighted data from the spreadsheet.

Opening the Run The Run Display is used to begin, view, interrupt, and abort data
Display collections in Auto Collect.

The Run Display contains the following elements:

• Status banner above the plots

• Two large plots
• Data acquisition counters
• Two pause options
To open the Run Display:
1. Apply settings to one or more wells (see Creating sample sets
for Auto Collect experiments (page 103)).
 Chapter 6: CSampler Plus data acquisition 111

2. Click OPEN RUN DISPLAY. The Run Display opens in the

main panel of the Auto Collect tab.

To close the Run Display and return to the Sample Annotation

table:
1. Click CLOSE RUN DISPLAY.
112 BD Accuri C6 Plus System User’s Guide

Running samples using the Auto Collect tab

Introduction This topic describes how to acquire data in the Auto Collect tab.

Before you begin • Set up plots and regions in the Manual Collect tab. You can
display two plots in the Auto Collect tab. See Creating a new
plot (page 157) and Creating and applying gates (page 160).

About collecting The BD CSampler Plus software begins data collection in the first
sample data well that has settings applied (most commonly A1) and progresses
horizontally or vertically as defined in the settings.

As data is acquired and the CSampler Plus advances through the

plate or tube rack, the software displays a small, blue square in the
upper left corner of each completed well.

A red border around a well indicates the sample that is currently

being acquired. The current well also flashes blue during data
acquisition.

Caution Data is collected into all wells with settings

applied, even if they already contain data.

Procedure To begin data acquisition using the Auto Collect tab:

1. Click the Auto Collect tab.

2. Select a plate type from the Plate Type menu.

 Chapter 6: CSampler Plus data acquisition 113

The software prompts you to save the workspace file.

The sample grid displays the available wells for data

collection, based on the assigned plate type:

– 96-well plate. Displays wells for rows A-H, columns 1-12

– 48-well plate. Displays wells for rows A-F, columns 1-8

– 24-tube rack. Displays wells for rows A-D, columns 1-6

Note: Changing the plate type automatically opens a new

blank workspace.

3. Name the plate by typing a label in the Plate Name field.

4. Define acquisition settings and create sample sets. See Creating

sample sets for Auto Collect experiments (page 103).

5. Create the Sample Annotation table. Creating the Sample

Annotation table (page 110).

6. Click OPEN RUN DISPLAY and select the plots to display.

7. Click AUTORUN. The status banner displays the current well

and the message Cytometer is running.

The Run Display also updates the acquisition counters and the
sample data in the plots.
114 BD Accuri C6 Plus System User’s Guide

8. When data collection is complete, DONE! is displayed

prominently across the Run Display.
 Chapter 6: CSampler Plus data acquisition 115

9. When a plate is complete, click CLOSE RUN DISPLAY to

close the Run Display and return to the Sample Annotation
table.

Stopping data To stop a run after it has started:

collection 1. Click on one of the following buttons:

• INTERRUPT PLATE completes acquisition for the current

well, then stops.
• ABORT WELL stops immediately before completing
acquisition for the current well. Restarts acquisition with
the next well.
116 BD Accuri C6 Plus System User’s Guide

To restart a run:
• To restart during an Interrupt plate state, click AUTORUN.
The software continues acquiring data where it left off.
• To restart a run during an Abort well state, do one of the
following:
– To begin collecting the sample in the next well, click
AUTORUN.
– To continue collecting from the same well, close the run
display on the Auto Collect tab, select the Manual Collect
tab and run the sample manually.

Viewing sample All plots created in the Manual Collect tab are listed in the Select
plots Plot list beneath the plot window. They can be selected at any time
during data collection. Two plots can be viewed at a time.

Plots can only be added, modified, or deleted from the Manual

Collect tab.

To view plots:
1. Select plots to view from the Select Plot list under each plot
corral.

More information • Creating a live gate (page 166)

 Chapter 6: CSampler Plus data acquisition 117

Ending a data acquisition session

Introduction This topic describes how to save the workspace file and clean the
SIP at the completion of a run.

Saving the To save the workspace file at the end of the run:
workspace file 1. Select File > Save.

Performing a SIP When you finish collecting samples, clean the SIP to ensure cells or
clean other particles are not left in the SIP. If you select Run SIP Clean
after samples at the bottom of the Control Panel, a SIP Clean will
be performed automatically at the end of the run.

To clean the SIP if it is not performed automatically at the end of

the run:
1. Click the Manual Collect tab.

2. Click SIP Clean.

A dialog opens prompting you to load tubes of BD FACSClean

and water.

3. Ensure the following tubes are in the designated locations on

the CSampler Plus. If necessary, click Eject Rack/Plate and
insert the tubes, then click Load Rack/Plate.

• 2 mL of BD FACSClean in the triangle location.

• 2 mL of DI water in the circle location.
118 BD Accuri C6 Plus System User’s Guide

4. Click SIP Clean.

More information • Creating a live gate (page 166)

 7
Data analysis
This chapter includes the following topics:

• Analyzing sample data (page 120)

• Setting up analysis plots (page 124)
• Creating an overlay histogram (page 127)
• Viewing data in plots from the Plot List (page 125)
• Statistics tab (page 130)
• Previewing a plot in the Statistics tab (page 132)
• Adjusting peak position with VirtualGain (page 134)
• Copying analysis data into other applications (page 142)
• Analyzing batches of samples (page 142)
• Compensation (page 146)
120 BD Accuri C6 Plus System User’s Guide

Analyzing sample data

Introduction This topic describes the Analyze tab and the controls and
indicators within the workspace.

About the Analyze The Analyze tab allows you to simultaneously view data from
tab multiple samples using the same plots and gating.

Use the tab to do the following:

• View several plots and samples in any combination for easy

analysis.
• Compare specific samples from the sample-well grid.
• Create new plots, hide or delete plots, and copy plots from the
Collect (or Manual Collect) tab.
• View different samples with the same plots.
• Create regions and markers.
• Create color overlay histograms.
• Print plots.
• Adjust peak position.
• Calculate median statistics.

Viewing the The Analyze tab is organized into two major sections:
Analyze tab
• Setup panel. Panel on the left side of the window that contains
controls for selecting samples and plots.

• Data display. Large area on the right side of the window that
shows sample data in plots and in a statistics table.

When you open the Analyze tab for the first time, the
workspace is empty. Plots can be copied from the Collect/
Manual Collect tab or created from scratch. Gating strategies
that were set up in the Collect (or Manual Collect) tab can be
applied in Analyze, or new gates can be drawn and new gating
strategies set up.
 Chapter 7: Data analysis 121

Analyze tab The following table describes each of the controls and indicators in
controls the Analyze tab:

Control Description
Plate Type Disabled in the Analyze tab. For the BD CSampler
Plus software only.

Plate Name Text box for naming the plate. For the
BD CSampler Plus software only.

Sample naming Text box for naming the current sample.

field
122 BD Accuri C6 Plus System User’s Guide

Control Description
Sample grid Matrix laid out in the configuration of a 96-well
plate to help organize sample data. Each sample
has its own well in the sample grid.
The wells are color-coded:
White. Does not contain data.
Blue. Contains data.
Black check mark. Currently selected for viewing
data.

Copy Plots from Copies specified plots from the Collect (or Manual
Collect Collect) tab.
See Copying plots from the Collect or Manual
Collect tab (page 124) for more information.

Make a new plot Buttons for creating new plots or overlaying

histograms.
See Creating a new plot (page 157) for more
information.

Plot List Lists the plots that are available in the Analyze
tab. Available plots include plots copied from the
Collect tab and plots created in the Analyze tab.

Set Color Opens the Color Compensation Matrix for

Compensation correcting fluorescence spillover.
See Correcting fluorescence spillover (page 149)
for more information.

Plot Corrals Area displaying two rows of plot corrals. Scroll up

or down to view more plots.

Statistics table Table below the plots that displays statistical

information for individual plots.
Statistics tables can be copied into most Microsoft
Office compatible programs.
 Chapter 7: Data analysis 123

Example data file An example data file of a four-color analysis of human peripheral
for analysis blood (HPB 4 Color Tutorial.c6) can be found on the BD Accuri
C6 Plus software installation disc. The data file can be used to
explore the various software analysis tools.

The example file consists of data from four sample tubes used to
assess the CD3+CD4+ and CD3+CD8+ cell populations. Samples
were prepared by staining peripheral blood with directly
conjugated antibodies, followed by red cell lysis, according to
standard preparation methods. The following table describes the
experimental design:

Tube 1. Background (unstained)

Tube 2. White blood cell gating control (CD45)

Tube 3. T-cell gating control

Tube 4. Experimental sample

Fluorochrome Tube 1 Tube 2 Tube 3 Tube 4

FITC Isotype Isotype CD3 CD3

PE Isotype Isotype Isotype CD4

PE-Cy7 Isotype CD45 CD45 CD45

APC Isotype Isotype Isotype CD8

This experimental design does not contain all single-stained

fluorescence controls, but only those required for determining
color compensation settings to correct fluorescence spillover. For
details on correcting fluorescence spillover, see Correcting
fluorescence spillover (page 149).
124 BD Accuri C6 Plus System User’s Guide

Setting up analysis plots

Introduction This topic describes how to create plots for analysis in the Analysis
tab.

Copying plots from In the Analyze workspace, you can copy plots from the Collect (or
the Collect or Manual Collect) tab or create new plots. Plots that are copied from
Manual Collect tab the Collect (or Manual Collect) tab are appended with a “C” (for
example, Plot 1C, Plot 2C).

If the plots you are copying contain regions and markers, the
regions and markers will copy over. The gating strategy does not
copy over.

To copy plots:
1. In the Analyze tab, click Copy Plots from Collect.

2. In the Copy Plots from Collect dialog, do one of the following:

• Select the checkboxes of the plots you want to copy.

• Select the All Plots checkbox to copy all of the plots from
the Collect tab.
 Chapter 7: Data analysis 125

3. Click OK. The selected plots are added to the Plot List.

4. To display the copied plots in the data display area, click an

empty plot corral, then click the plot from the Plot List.

5. Click the sample well that contains the data you want to view.
For more information on viewing the data, see Viewing data in
plots from the Plot List (page 125).

6. Repeat as necessary to display all plots.

Viewing data in To view a plot in the Plot List:

plots from the Plot 1. Click on an empty plot corral in the Analyze tab.
List
2. Click a plot in the Plot List to open the plot. The plot is
displayed without any sample data.

Any gates copied from the Collect tab are renamed (for
example, P1 in Collect is P2 in Analyze). These gates can be
adjusted in the Analyze tab without changing the position of
the gates in the Collect tab.

3. Click the well of the sample you want to view.

126 BD Accuri C6 Plus System User’s Guide

4. Apply a gating strategy, if desired. See Creating and applying

gates (page 160).

5. To view data from another sample, open one or more plots

from the Plot List (we recommend starting another row of
plots). Click a plot, then choose the sample to be displayed in
each plot. The gates that you applied above are automatically
applied to the corresponding plots in the new row.

6. Compare data and statistics between samples.

 Chapter 7: Data analysis 127

Creating new plots To create a new plot:

1. Click an empty plot corral.

2. Click one of the following Make a new plot icons:

• Histogram
• Dot
• Density
• Overlay Histogram
An FSC-A histogram or FSC-A vs SSC-A dot or density plot is
displayed.

3. Click the sample well that contains the data you want to view.

More information • Creating and applying gates (page 160)

Creating an overlay histogram

Introduction This topic describes how to create an overlay histogram. You can
compare multiple distributions from different samples at the same
time by creating an overlay histogram.

Procedure To create an overlay histogram:

1. Click an empty plot corral.
128 BD Accuri C6 Plus System User’s Guide

2. Click the Overlay Histogram tool to open a blank single-

parameter FSC-A plot.

3. Click the x-axis label (FSC-A) and select a different parameter,

if desired.

4. Click GATE and apply a gate, as appropriate.

5. Select the data wells to be overlaid from the 96-well grid.

 Chapter 7: Data analysis 129

6. Click the Overlay Histogram Legend tool below the plot to

view a legend for the overlay histogram.

7. (Optional) To change the color of one or more overlay plots,

click the square in the Overlay Histogram Legend and select
the desired color from the color palette. Click OK.
130 BD Accuri C6 Plus System User’s Guide

Statistics tab
Introduction This topic describes the Statistics tab and how to create custom
statistics.

About the Statistics The Statistics tab provides a way of tabulating data from multiple
tab samples in one master table. It also allows you to do the following:

• View statistics for some or all of your collected samples.

• Display statistics of collected or imported samples.
• List all plots created on the Collect (or Manual Collect) and
Analyze tabs.
• Display all plot names, gates, and associated statistics.
• Cut and paste data into a spreadsheet.

Viewing the The Statistics tab is organized into two major sections:
Statistics tab
• Setup controls (Statistics Column Selector, Sample Selector,
and Display Plot Preview) for the Master Statistics table
• Master Statistics table
When the Statistics tab is first opened, the workspace is empty.

To create a Master Statistics table, select statistics from the

Statistics Column Selector and samples from the Sample Selector.
See Creating the Master Statistics table (page 131) for more
information on creating the Master Statistics table.
 Chapter 7: Data analysis 131

Statistics tab The following table describes each of the controls and indicators in
controls the Statistics tab:

Control Description
Plot Previews the selected plot.

Display Plot List of plots imported from the Collect and

Preview Analyze tabs that allows you to select the plot
to preview.

Statistics Column Allows you to select the data to view in the

Selector Master Statistics table for each sample.

Sample Selector Allows you to select the samples to view in the

Master Statistics table.

Master Statistics Configurable table that displays data of

table selected samples. See Creating the Master
Statistics table (page 131).

Creating the The Master Statistics table allows you to immediately view selected
Master Statistics data across multiple samples. You can customize which data you
table want to view for each plot, and you can modify the table at any
time.

To create the Master Statistics table:

1. In the Statistics Column Selector, select the checkboxes under
the data items you want to view per plot. Use the Display Plot
Preview to help decide which statistics to select.

Stats are added in the order they are selected after they are
grouped by plot and region.

The software automatically adds the corresponding columns

to the Master Statistics table.
132 BD Accuri C6 Plus System User’s Guide

2. In the Sample Selector list, select the checkbox of each sample

you want to view. The software automatically adds rows of
samples to the Master Statistics table and displays the sample
data.

More information • Previewing a plot in the Statistics tab (page 132)

Previewing a plot in the Statistics tab

Introduction This topic describes how to preview a plot in the Statistics tab.
 Chapter 7: Data analysis 133

Procedure To preview a plot in the Statistics tab:

1. In the Display Plot Preview list, click the plot you want to
preview.

2. In the Sample Selector list, select the Preview button of a

sample. The software displays the sample’s data in the plot.

The plot preview is available for viewing only. You can modify
the zoom level and other plot settings in the Analyze or Collect
tab.
134 BD Accuri C6 Plus System User’s Guide

More information • Statistics tab (page 130)

Adjusting peak position with VirtualGain

Introduction This topic explains VirtualGain™ software and how it can be used
to mimic voltage and amp gain adjustments to reposition data on
the axis after the data has been collected.

About VirtualGain In certain instances, a particular peak should have the same
position across different samples or be located at a specific channel
number, regardless of the staining. Instruments that have voltage
and amp gain controls allow you to adjust peak position from
sample to sample. BD CSampler Plus software uses VirtualGain
instead of these controls.

VirtualGain is a software module that mimics voltage and amp

gain adjustments to reposition data on the axis after the data has
been collected. VirtualGain makes gross adjustments (approximate
visual shifts of the data) of histogram plots. It is strictly an analysis
tool and should not be used while collecting data.

For example, in the following figure, the negative peaks in the

control sample and in sample 1 fall in similar channels (mean value
= 28.2 and 29.7, respectively). However, the negative population in
sample 2 is farther to the right (mean value = 73.4). You can use
VirtualGain to align the negative peak of sample 2 with the control
sample.
 Chapter 7: Data analysis 135

Applying Only apply VirtualGain on a histogram plot, and only on one

VirtualGain parameter at a time. After VirtualGain is applied, you can view
data in any type of plot and toggle VirtualGain on and off.

VirtualGain is only applied to the displayed data and does not alter
FCS data. The adjustment is recorded only in the data file.

To apply VirtualGain to plots:

1. In the Analyze tab, do one of the following:

• Recreate the histogram to which you want to apply

VirtualGain.
• Copy plots from the Collect tab.
136 BD Accuri C6 Plus System User’s Guide

2. Apply the appropriate gating to the plots in the Analyze tab.

3. Do one of the following:

• Select a histogram plot from the sample to which the other

samples will be aligned. This sample is the standard
sample.
• Select an empty well if you want to align data to a specific
channel instead of a collected sample.
4. Click the x-axis label on the standard sample and select
VirtualGain.

5. In the VirtualGain dialog, do one of the following:

• Move the peak definition marker (vertical line) in the

standard sample plot to the center of the peak that will be
the reference point. Other samples will be aligned to this
position.
 Chapter 7: Data analysis 137

• If you selected an empty well in step 3, move the peak

definition marker (vertical line) to the channel that you
want to assign as the reference point.

6. If needed, use the Zoom tools in the Analyze tab to change the
zoom level in the VirtualGain dialog.

7. Click the small sample grid icon in the center of the Sample to
Align plot.

8. Open the sample to be aligned by clicking the corresponding

blue well in the sample grid (the gray well indicates the
138 BD Accuri C6 Plus System User’s Guide

standard sample currently selected). Ensure that this plot has

been zoomed to the required level before setting VirtualGain.
 Chapter 7: Data analysis 139

9. Move the peak definition marker in the Sample to Align plot

to the center of the peak that you want to align.

10. Click Preview to view the aligned sample with VirtualGain

applied.

VirtualGain aligns the peak of interest in both plots.

11. Repeat steps 9 and 10 to make additional adjustments, if

needed.

12. To align additional samples exactly as the first aligned sample,

select This sample and then click the well(s) in the sample grid
that you want to include.

If the other samples need a different amount of VirtualGain,

set VirtualGain separately for each one.

13. Click Apply to apply VirtualGain to the data.

140 BD Accuri C6 Plus System User’s Guide

The software displays a black asterisk under the Sample to

Align plot to indicate that VirtualGain has been applied to the
specified parameter for that sample.

14. Click Close to close the VirtualGain dialog.

Viewing When VirtualGain is applied to a sample, an asterisk is displayed

VirtualGain under the parameter label in the associated plot.

The asterisk is color-coded:

• Black. VirtualGain has been applied.

• Gray. The software is currently displaying the original data.

Overlays automatically display VirtualGain when it is applied. The

asterisk is not displayed in overlays when VirtualGain is applied to
some or all of the samples in the overlay.
 Chapter 7: Data analysis 141

Changing To toggle between views with VirtualGain applied and not

VirtualGain views applied:
1. Click the asterisk in the plot.
142 BD Accuri C6 Plus System User’s Guide

Removing You cannot undo this action.

VirtualGain
To permanently remove VirtualGain from every parameter in
every sample in the data file:
1. Select Display > Remove All VirtualGain.

Copying analysis data into other applications

Introduction This topic describes how to copy analysis data from the Master
Statistics table into most Microsoft Office compatible applications.

Procedure To copy data:

1. Use the mouse to highlight the fields you want to copy.

2. Press Ctrl+C to copy the data.

3. In the Microsoft application, press Ctrl+V to paste the data.

More information • Copying and printing plot data (page 181)

Analyzing batches of samples

Introduction This topic describes how to analyze batches of samples.

About batch The Batch Analysis tab allows an automated analysis to be

analysis performed on multiple samples at the same time. The plot types
created during acquisition will appear at the top of the screen.

Statistics can be displayed for individual files or all selected files.

The statistics displayed are default stats and can not be
customized. After analysis, regions can be moved or resized on
 Chapter 7: Data analysis 143

plots for individual samples without affecting the plots for other
samples.

Viewing the Batch The Batch Analysis tab is organized into two major sections:
Analysis tab
• Setup panel. Panel at the top that contains the sample grid and
available plots for selecting samples and plots.

• Data display. Large area at the bottom that shows sample data
in plots and in a Statistics table.

When the Batch Analysis tab is opened for the first time, the
workspace is empty. To set up the tab, plots can be created or
copied from the Collect (or Manual Collect) or Analyze tab.
144 BD Accuri C6 Plus System User’s Guide

The following table describes each of the controls and indicators in

the Batch Analysis tab:

Control Description
Sample grid Matrix laid out in the configuration of a 96-well
plate for selecting the samples to analyze.
The wells are color-coded:
White. Does not contain data.
Blue. Contains data.
Black check mark. Currently selected for batch
analysis.

Available plots Displays plots (without data) that can be included

in the batch analysis. Available plots include plots
copied from the Collect tab or created in the
Analyze tab.

Analysis pane Rows of analysis data in table and plot format,

based on the plots that were selected for use in
analysis.
Statistics tables can be copied into most Microsoft
Office compatible programs.

Running a batch To run a batch analysis:

analysis 1. At the completion of a sample run, click the Batch Analysis
tab.

2. Do one or both of the following:

• Select the Show Plots from Collect checkbox to make all of

the plots from the Collect tab available for batch analysis.
• Select the Show Plots from Analyze checkbox to make all
of the plots from the Analyze tab available for batch
analysis.
3. Select the specific plots to include in the batch analysis by
selecting the checkboxes under those plots.
 Chapter 7: Data analysis 145

4. Select the samples to analyze in the sample grid by clicking on

the wells.

One row is created for each sample.

5. To show the statistics associated with samples, do one of the

following:

• Select the Show Statistics with All Plots checkbox to show

statistics in every row.
• Select the Show Statistics checkbox in individual rows to
view statistics for specific samples. Regions can be moved
and resized.

Exporting data Plots and statistics can be exported in two ways, either as a
Microsoft PowerPoint® file or as a Microsoft Excel® spreadsheet.

To export data:
1. Do one of the following in the lower right-hand corner of the
Batch Analysis tab:

• Click Export to PowerPoint.

• Click Export Stats to Excel.

2. When exporting to PowerPoint, you can choose to export the

statistics tables with the plots, then select where you want
them to appear.
146 BD Accuri C6 Plus System User’s Guide

3. In the Save dialog, navigate to the destination folder, type a file

name, and click Save.

More information • Renaming plots (page 160)

• Renaming regions (page 174)
• Applying color gating (page 172)

Compensation
Introduction This topic explains the concept of fluorescence spillover, how to
recognize when spillover occurs, and how to correct it using
compensation.

About fluorescence Fluorochromes typically emit light over a broad range of

spillover wavelengths, resulting in the fluorescence signal appearing not
only in the expected, primary cytometer detector, but in other
detectors as well.

This phenomenon is often called fluorescence spillover, and can be

a source of confusion when interpreting multicolor flow cytometric
data.

You can remove fluorescence spillover from plots by applying a

mathematical algorithm to the data. This process is referred to as
color compensation. The color compensation algorithm subtracts a
percentage of fluorescence signal from every event, removing the
fluorescence spillover. Because data collection on the cytometer is
digital, color compensation can be applied or removed before,
during, or after data collection.

Recognizing The following figure shows data collected for a PE-Cy™7 single-
fluorescence stained control. Most of the fluorescence signal from PE-Cy7–
spillover positive cells was detected in FL3 (670 LP), as expected.
 Chapter 7: Data analysis 147

However, there is also PE-Cy7 signal detected in FL1 (533 BP) and
FL2 (585 BP), so that data plots for those detectors appear to have
positively fluorescing cells. No signal from PE-Cy7 appears in
detector FL4.

Compensation Compensation window

controls The compensation controls in software either automatically
remove (subtract) or allow you to manually remove the spillover
from the non-primary detectors. You can use default settings
obtained from Instrument QC, or manually enter the percentage of
the signal you want to subtract from a detector. The compensation
window works together with the compensation preferences. See
Compensation preferences (page 148). The Well Preference at the
top of the window, shows the compensation preference setting for
the selected well when it was acquired.

The Reset to BD Defaults button appears when the compensation

preference is set to From Instrument QC. If you modify the settings
148 BD Accuri C6 Plus System User’s Guide

for a well acquired using the default compensation from

Instrument QC, you can use the Reset button to revert to the
default QC compensation settings.

Use the Apply to all samples checkbox to apply the compensation

settings for the selected well to all other wells, regardless of the
collection status. Click the well, select Apply to all samples, then
click Apply. If you wish to reset the compensation settings to the
BD default values, only the selected sample will be set to the
default settings; not all samples to which the settings were applied.

Compensation preferences
The Preferences button in the compensation window allows you to
select between using your own custom user-defined compensation
settings or the compensation settings set by the software during
Instrument QC using BD CS&T RUO beads. Instrument QC sets
compensation for FITC, PE, PerCP, PerCP-Cy™5.5, and APC. If
you choose to use the settings from Instrument QC, you can select
to use FL3 compensation values for either PerCP or PerCP-Cy5.5.
If you use fluorochromes other than those set by instrument QC,
compensation will not be set for those fluorochromes. You will
need to manually adjust compensation for other fluorochromes.
 Chapter 7: Data analysis 149

The option you select (either User Defined or From Instrument

QC) becomes the default setting for all empty wells in the
workspace. However, even if you select From Instrument QC as
the default, you can still manually adjust compensation values for
individual samples.

When saving a workspace template, if the compensation preference

is set to User Defined, the user-defined compensation values will be
saved with the template. If the preference is set to From Instrument
QC, the software will use the QC values that are current when the
sample is acquired.

Correcting When color compensation has been properly applied to a data set,
fluorescence the median fluorescence channel value in non-primary detectors
spillover for any given single-stained control sample should be the same as
that of an unstained control sample.

Whenever performing a multi-color experiment where manual

compensation adjustment is required, prepare a set of control
samples, each stained with one individual fluorochrome. These
single-stained controls will allow you to determine the extent of
fluorescence spillover from each fluorochrome.
150 BD Accuri C6 Plus System User’s Guide

To correct fluorescence spillover:

1. Add quadrant markers to the plot you want to correct by
clicking the Quadrant tool then clicking inside the plot.

2. Adjust the quadrant marker position so that all positive

populations are contained within individual quadrants.

The median fluorescence channel value for the events in each

quadrant is displayed in the Statistics table (shown below). No
median is calculated for the first line named All or This Plot
because this line contains summary data for the entire plot.

3. Compare the median values of the affected channel. If the

median value of the UL or LR quadrant is not equal to the
median value of the negative population (LL), you will need to
adjust fluorescence compensation.

4. Click Set Color Compensation in the Collect or Manual

Collect tab or select Instrument > Set compensation in the
 Chapter 7: Data analysis 151

Analyze tab to open the Compensation Settings window. The

window contains four rows—one for each fluorescence
channel.

Note: You cannot change the compensation setting for

individual samples from the Auto Collect tab. However, you
can change the compensation settings for any sample during
analysis from the Analyze tab.

5. Do one of the following:

• In the appropriate % text box, type an arbitrary percentage

of the signal to subtract.
• Use the Compensation Calculator to calculate the
subtraction values. The Compensation Calculator is an
Excel file that you can use to determine compensation
values, especially for fluorochromes not calculated during
instrument QC. Go to bdbiosciences.com, navigate to the
BD Accuri C6 Plus cell analyzer, and select Resources. The
compensation calculator can be found under Tools.
6. Click Preview to update the Statistics table.

7. In the Statistics table, observe the values in the Median column

of the FL channel you are correcting.
152 BD Accuri C6 Plus System User’s Guide

8. Repeat steps 5-8 until the median value for the UL or LR

quadrant is equal (or nearly equal) to the median value of the
negative population (LL).

This value is called the compensation value. The figure below

shows the median values highlighted in blue.

The following figure shows the data after proper color

compensation has been applied.
 Chapter 7: Data analysis 153

9. If you want to apply the fluorescence subtraction to all

samples, select Apply to all samples in the Compensation
Settings window, then click Apply.
154 BD Accuri C6 Plus System User’s Guide
 8
Plots and gates
This chapter covers the following topics:

• Plots and gates (page 156)

• Creating a new plot (page 157)
• Creating and applying gates (page 160)
• Creating nested gates (page 167)
• Modifying plots (page 175)
• Zooming a plot (page 179)
• Copying and printing plot data (page 181)
156 BD Accuri C6 Plus System User’s Guide

Plots and gates

Introduction This topic describes plots and the tools used to create different
types of plots, gates, and markers.

About plots Plots allow you to view sample data in histogram, density, dot, and
overlay histogram plots. You can view multiple plots for each
sample you collect.

Plot tools Each plot contains a set of gating and marking tools and a set of
viewing tools.

Gating and marking tools include:

• Gate button. Opens the Change Gating dialog for applying
gates to a plot.

• Polygonal gating tool. Allows you to draw a polygon around a

population of events.

• Rectangular gating tool. Allows you to draw a rectangle

around a population of events.

• Elliptical gating tool. Allows you to draw an ellipse around a

population of events.
 Chapter 8: Plots and gates 157

• Quadrant markers tool. Allows you to draw quadrants in a

plot to partition the plot into four sections.

• Hinged markers tool. Allows you to draw hinged markers in a

plot to partition the plot into four sections. Similar to
quadrant markers but with more flexibility.

• Vertical marker tool. Used for gating histograms to the right or

left of a vertical marker. Appears for histogram plots only.

• Horizontal marker tool. Used for gating histograms within a

horizontal marker. Appears for histogram plots only.

Plot viewing tools include:

• Plot Spec tool. Opens the Set Plot Specs dialog for changing
the x- and y-axis parameters, scaling the plot, and setting log
or linear view.

• Zoom tool. Allows you to select a rectangular region in the

plot to zoom in on.

• Expand tool. Zooms out one level with each click of the tool.

Creating a new plot

Introduction This topic describes how to create a new plot.

Procedure To create a new plot:

1. Click on one of the following icons in an empty plot corral:

• Histogram Plot
• Dot Plot
• Density Plot
158 BD Accuri C6 Plus System User’s Guide

Note: Overlay histogram plots are available from the Analyze

tab during analysis. See Creating an overlay histogram
(page 127).

The software displays an FSC-A vs SSC-A plot (or FSC-A, for

histograms) by default.

2. Configure the plot specifications as needed.

Changing plot The Plot Spec tool allows you to change the way data are displayed
specifications in a plot. You can change the axis parameter, specify channel
ranges, and toggle between linear and logarithmic scales. The Plot
Spec tool is available in the Collect and Analyze tabs.

You can set up or modify plot specifications at any time before or

after collecting data.
 Chapter 8: Plots and gates 159

To change the plot specifications:

1. Click the Plot Spec tool.

2. In the Set Plot Specs dialog, do the following for each axis:

• Select the parameter(s) to view from the X/Y-AXIS list(s).

• Select linear or log to specify how data are displayed.
• Type in the minimum and maximum channels to set the
channel range you want to view.
• Select or clear the Hide 1st decade checkbox to indicate
whether you want the software to display the first decade
of channels (channels 1–10) in the plot for log data.
3. Do one of the following:

• Click Apply to apply the changes without closing the

dialog.
• Click OK to apply the changes and close the dialog.
• Click Cancel to close the dialog without applying the
changes.
160 BD Accuri C6 Plus System User’s Guide

Renaming plots Plots can be renamed. By default, plots are named after the order
in which the plot was created and the associate plate map well, for
example Plot 1: A01.

To rename a plot:
1. Double-click the plot name.

2. In the text field, type in a new name.

3. Press Enter.

The well number will remain in the new plot name, for
example, LWB: A01. The new name will apply to the selected
plot for all samples in the run, for example, LWB: A02.

The plot name is also updated in the plot name column

heading of the statistics table.

More information • Creating an overlay histogram (page 127)

Creating and applying gates

Introduction This topic describes how to create and apply gates, including a live
gate used during acquisition.

About gates A gate is a specified area that is used to designate a population of

events to analyze. You can create any of the following types of
gates.

• Polygonal gate. An irregularly shaped polygon area, with an

unlimited number of segments, around a population of events.
 Chapter 8: Plots and gates 161

• Rectangular gate. A rectangular area.

• Elliptical gate. An elliptical area.

• Quadrant gate. Quadrants to create four partitions within a

plot.

• Hinged quadrants. Quadrant markers with a hinge at the

intersection.

• Vertical marker. Gates a histogram plot to the right or left of a

vertical marker.

• Horizontal marker. Gates a histogram plot within a horizontal

marker.

Creating a gate in a To create a gate in a density or dot plot:

density or dot plot 1. Click on one of the following gating tools:

• Polygon
• Rectangle
• Ellipse
• Quadrants
• Hinged quadrants
2. Use the mouse to draw a region (labeled P1 for polygon, R1
for rectangle, E1 for ellipse, Q1 for quadrants, and H1 for
hinged quadrants).

If you are drawing a polygon, click the mouse to anchor each

vertex and double-click to close the polygon.
162 BD Accuri C6 Plus System User’s Guide

The software automatically displays the percentage of cells

within the gate.

3. (Optional) You can change the gate labels by double-clicking

on the label and typing a new gate name in the text box.

Creating a vertical You can create a single vertical marker per histogram to obtain
marker in a statistics on the events to the left and right of the marker.
histogram plot
To create a vertical marker in a histogram plot:
1. Click the Vertical Marker tool.

2. Click the cursor at the point along the x-axis where you want
to place the marker.
 Chapter 8: Plots and gates 163

The percentage of cells is automatically displayed to the left

(V1-L) and right (V1-R) of the marker.

You can create only one vertical marker per histogram.

However, you can create one or more horizontal markers
within the same histogram.

Creating a You can create a horizontal marker within a histogram to obtain

horizontal marker statistics on the events within the boundaries of the marker.
in a histogram plot
To create a horizontal marker in a histogram plot:
1. Click the Horizontal Marker tool.

2. Drag the cursor horizontally across the area you want to gate.
164 BD Accuri C6 Plus System User’s Guide

The percentage of cells is automatically displayed within the

boundaries of the marker (labeled M1).

You can create multiple horizontal markers in a histogram

plot.

Deleting a gate To delete a gate:

1. Click to select the gate.

2. Press the Backspace key on the keyboard.

If the gate is being used in other plots, a message appears

warning you that the gating in other plots will be removed.

Applying a gate to To apply a gate to a plot:

a plot 1. Click GATE at the top of the plot for which you want the gate
applied.

The Change Gating dialog appears.

2. Only polygon (P), rectangular (R), elliptical (E), hinged

quadrants (H), and marker (M) gates automatically appear in
the Change Gating dialog list of options. If you want to view a
 Chapter 8: Plots and gates 165

list of vertical markers (V) or quadrant markers (Q), select the

associated checkbox(es).

If the plot to which you are applying a gate already has a gate,
it will not appear in the list. Only gates in other plots appear.

3. Select one of the following gating icons to the left of the region
in the list.

– Include. To analyze the events within the region. You can

choose more than one gate with Include to analyze events
in either one or the other gate.

– Exclude. To analyze the events outside of the region.

– Intersection. To analyze the events within the intersection

of two or more regions. Select this icon for each region you
want to use.
166 BD Accuri C6 Plus System User’s Guide

4. Click Apply.

The software displays the type of gate that is applied next to

the GATE button in the plot.

Creating a live gate Live gating is used to exclude events outside a designated region
from being displayed and stored as part of the workspace or FCS
file. Any region may be used as a live gate. This selection can be
remembered as part of the template if desired.

Once a live gate is used for data acquisition, there is no way of

recapturing the excluded data that was not acquired.

To create a live gate:

1. Create a region around the population of interest (the
population you want to collect).

2. Select the Do not collect events outside checkbox.

Note: You can run unlimited or with limits.

3. Select the region from the list (for example, R1).

 Chapter 8: Plots and gates 167

Only the events that appear in the selected region will be

acquired.

More information • Creating nested gates (page 167)

• Modifying regions (page 171)

Creating nested gates

Introduction This topic explains nested gates and describes how to create and
apply them within a plot.

About nested gates You can create a series of nested gates in which each gate is a
subset of the previous one. This allows you to fine tune your gating
strategy for viewing and analyzing specific subsets of data.
168 BD Accuri C6 Plus System User’s Guide

Creating nested To create nested gates:

gates 1. Draw any region or marker around a population of events.

2. Click GATE in a second plot and apply the gate. See Applying
a gate to a plot (page 164). This is the parent gate.
 Chapter 8: Plots and gates 169

3. Close the dialog. The plot displays only the populations within
the parent gate.

4. In the plot that is gated on the parent gate, draw a second

region or marker around a subset of the population displayed
in the plot.

5. Open a third plot and click GATE.

170 BD Accuri C6 Plus System User’s Guide

6. In the Change Gating dialog, select the option in which the

second gate is “in” the parent gate (see the following figure).
Do not select the on all events option.

7. Apply the gate. This is the child gate.

8. View the statistics in the Statistics table.

9. Close the dialog.

 Chapter 8: Plots and gates 171

More information • Creating and applying gates (page 160)

• Modifying regions (page 171)

Modifying regions
Introduction This topic describes how to modify regions by moving and resizing
them and coloring the events in a region.

Moving and You can move or resize a region. You can also move a region label
resizing regions by clicking and dragging the label.

To move a region:
1. Click the border of the region. The outline appears in bold.
172 BD Accuri C6 Plus System User’s Guide

2. Position the cursor on the border, but not an anchor point

until a four-sided arrow appears, then drag the region to the
desired position.

To resize a region:
1. Click the border of the region. The outline appears in bold.

2. Position the cursor on one of the anchor points until a two-

sided arrow appears, then drag the region to resize it.

To rotate an ellipse region:

1. Click the border of the region. The outline appears in bold.

2. Position the cursor on one of the anchor points until a two-

sided curved arrow appears, then drag the region to rotate it.

Applying color All events within one or more regions can be designated to appear
gating as a specified color within other histogram or dot plots. The event
coloring will remain and automatically update during data
acquisition.

Color will not appear in a density plot.

 Chapter 8: Plots and gates 173

To assign a color to the events in a gate:

1. Create a region in a histogram, dot, or density plot.

2. Double-click the region name and click the small white square
to display a color palette.

The most commonly used colors appear on the top row.

3. Select a color.
174 BD Accuri C6 Plus System User’s Guide

Events that fall within the gate are now colored and displayed
in the same color in other dot and histogram plots.

Renaming regions Regions in a plot can be renamed.

To rename a region:
1. Double-click the region name.

2. In the text field, type in a new name and press Enter.

 Chapter 8: Plots and gates 175

Modifying plots
Introduction This topic describes how you can change the number of events in a
plot and change the plot parameters and axes names.

Changing the You can change the number of events displayed in all plots across
number of events all samples to make it easier to view the data. This option allows
in a plot you to visually remove a number of events from the plot without
deleting data.

To change the number of events displayed in a plot:

1. Select Display > Events Display Settings.

2. Select the events to display:

• To view all collected events, select Show all events.

• To view the first N events of a sample, select Display first
and type a number in the Events Collected field.
• To view a specified percentage of the whole in a pseudo-
random selection, select Display and type a percentage to
176 BD Accuri C6 Plus System User’s Guide

view (for example, if 20% is selected, every fifth event is

displayed).

3. Click Apply to apply your settings without closing the dialog.

Or, click OK to apply your settings and close the dialog.

The software displays the plot and shows a message in the plot
that some events are not being displayed.

Changing plot You can change the parameters that a plot displays for the x- and
parameters y-axes. By default, the software displays the area parameter
(signified with a suffix of -A), but you can also choose a height,
width, or time parameter.
 Chapter 8: Plots and gates 177

To change a parameter:
1. Click the x- or y-axis label and select the option you want
from the menu.

Note: The time parameter, which can be displayed on the

x-axis only, starts counting when you click RUN and
continues counting for 19 days (16 million tenths of a second),
even if you add data to the sample at a later time. You cannot
reset the parameter to zero, even by deleting data.

Renaming plot axes You can rename the axis labels for a plot to identify the antibody
staining or fluorochrome used in the sample.

To rename a plot axis:

1. Click on an axis label and select Rename Parameters/Color
Compensation from the menu.
178 BD Accuri C6 Plus System User’s Guide

2. In the Compensation Settings dialog, type the new label in the

text box for the parameter you want to rename.

3. Do one of the following:

• Click Apply to apply the label to the selected sample.

• Select Apply to all samples, then click Apply to apply the
label to all samples.
 Chapter 8: Plots and gates 179

Zooming a plot
Introduction This topic explains the zoom function you can use to zoom in on
specific areas of a plot.

Zooming on a To zoom on a population:

population 1. Click the Zoom tool.

2. Drag the mouse in the plot to draw an area to zoom.

The following figure shows a population before and after

using the Zoom tool.

Before zoom After zoom

3. Repeat the zoom as needed to zoom in closer.

Zooming out To zoom out:

1. Click the Expand tool to the right of the zoom tool.

2. Repeat as needed.
180 BD Accuri C6 Plus System User’s Guide

Zooming to a Sometimes it can be helpful to view the data within a specified

specified channel channel range.
range
To view a specified channel range in a plot:
1. Click the Plot Spec tool in the plot you want to zoom.

2. Specify the x-axis channel range by typing a minimum (Min

Value) and maximum (Max Value) value under X-AXIS in the
Set Plot Specs dialog.

3. Specify the y-axis channel range by typing a minimum and

maximum value under Y-AXIS.

4. Click Apply to apply the changes and click OK to close the

Plot Spec dialog.
 Chapter 8: Plots and gates 181

Copying and printing plot data

Introduction This topic describes how to copy and paste plots into third-party
software and how to print plot data.

Copying and To copy and paste plots from the Collect or Analyze tab:
pasting plots 1. Click anywhere on a plot and drag it to an open Microsoft®
Office compatible application.

You cannot use Ctrl+C and Ctrl+V to copy and paste plots
from the software into other applications.

For information on copying and pasting plots from the Collect tab
to the Analyze tab, see Copying plots from the Collect or Manual
Collect tab (page 124).

Selecting a drag You can select one of two file formats for plots during drag and
and drop format drop actions.

To select the file format to use:

1. Select File > Set Plot Drag and Drop Format.

2. Choose one of the following formats:

• Select .png when lower resolution is sufficient. Drag and

drop to Excel requires .png format.
• Select .eps when higher resolution images are required for
publication or posters.
182 BD Accuri C6 Plus System User’s Guide

The Drag and Drop Format selection is file-specific, and will

always revert to the default of .png when a new C6 Plus file is
created or if the current file is not saved with the .eps option
selected.

3. Click OK to save the settings and return to the workspace.

Printing plots and You can print selected plots and their associated statistics from the
statistics Collect or Analyze tab.

To print plot data:

1. Select the checkbox in the upper-left corner of each plot that
you want to print.

2. If you do not want to print the associated Statistics tables,

clear the checkbox in the upper-left corner of each table.

3. Select File > Print Selected Items.

More information • Copying plots from the Collect or Manual Collect tab
(page 124)
• Copying analysis data into other applications (page 142)
 9
Managing data
This chapter covers the following topics:

• Managing files (page 184)

• Workspace files (page 186)
• Creating a workspace template (page 189)
• Exporting and importing data (page 190)
184 BD Accuri C6 Plus System User’s Guide

Managing files
Introduction This topic describes the files generated by BD Accuri C6 Plus
software.

About C6 Plus files The BD Accuri C6 Plus system generates the following files.

File Description
FCS FCS 3.1 files are not saved by default, but can be
exported at any time following acquisition. See
Exporting and importing data (page 190) for
information.

Workspace Workspace files contain the entire workspace and all

elements associated with the workspace, including the
sample data. See Workspace files (page 186) for more
information. Workspace files are saved to the location
you select when prompted to save the file.

Results CSV CSV files are saved only if you select to save them. A
single CSV file is saved for each sample. Files are
saved to the location you choose. See Exporting and
importing data (page 190) for more information.

QC reports A QC report is saved for each instrument QC test.

Reports are saved to C:\Cytometer Support
Files\Instrument QC reports.

Note: Levey-Jennings reports can be printed, but they cannot be

saved or exported.

Removing QC You can remove old QC runs that appear in the history file. See
report files from Viewing previous QC results (page 61).
the previous QC list
1. Navigate to C:\Cytometer Support
Files\InstrumentPerformance.

Each QC results file is listed as InstrumentPerformanceyyyy-

mm-dd-hh_mm.ipr.
 Chapter 9: Managing data 185

2. Delete the QC results files you no longer need.

The QC results for the dates you deleted will no longer appear
in the history file list. However, the results will still appear in
the Levey-Jennings plots.

Removing bead lots You can remove old CS&T RUO bead lots so that they no longer
from the CS&T appear in the bead lot menu on the QC workspace.
bead lot menu
1. Navigate to C:\Cytometer Support
Files\InstrumentPerformance\BeadLots.

Bead lot files have the extension .bls.

2. Delete the bead lot files you no longer need.

The deleted bead lots will no longer appear in the BD CS&T

Bead Lot menu on the QC workspace. If you need to replace a
deleted bead lot, do not copy the file into this folder. You must
reinstall bead lots using the software. See Install a new bead lot
(page 60).
186 BD Accuri C6 Plus System User’s Guide

Workspace files
Introduction This topic describes how to save the data to a workspace file.

About saving Always save data as a workspace file (.ci). A workspace file is a
workspace files comprehensive (and often large) file that contains instrument
settings, FCS files, and plot layouts.

The file contains the entire workspace, including the following

elements:

• Sample data
• Plot layouts
• Parameter names
• Zoom levels
• Gating
• Color compensation
• Threshold settings
• Collect (or Manual Collect) tab settings
• Changes made in the Analysis or Statistics tab
When a file is saved, the software displays the file name in the
upper-left corner of the workspace.

Auto-saving data By default, the software automatically saves the event data any
time the cytometer reaches a run limit or if you click Pause during
a run. Auto-save does not save changes to acquisition settings,
plots, or gating strategies that occur after the initial save when
naming the file.
 Chapter 9: Managing data 187

To save the entire workspace file, save the file manually.

Caution If you make changes after a run or after

pausing a run, the software does not automatically save
the file. Save these changes manually.
To enable or disable auto-save:
1. Select File > Auto-save Data Settings.

2. Do one of the following in the Auto-save Data Settings dialog:

• Select Auto-save Data Enabled to enable auto-save.

• Select Auto-save Data Disabled to disable auto-save.

3. Click OK.

4. If prompted to save the workspace before closing, do one of

the following:

• Click Yes to save the entire workspace.

• Click No to exit the dialog without saving the most recent
changes.

Manually saving You can manually save a workspace file at any time.
files
Note: For BD CSampler Plus software, you will be prompted to
save the workspace file after you select the plate type.

To manually save a workspace file:

1. Select File > Save.
188 BD Accuri C6 Plus System User’s Guide

If this is the first time the file is being saved, a save dialog
appears. Enter a file name and click Save.

To save a workspace file with a new name:

1. Select File > Save workspace as.

2. Navigate to the location where you want to save the file, enter
the file name, and click Save. The file is saved with the
extension .ci.

Opening a new A new workspace displays a single FSC-A versus SSC-A density
workspace plot that is pre-zoomed to channel values of 1,600,000 and
800,000, respectively. Run settings are set to Run Unlimited, the
primary threshold is set to channel 80,000 on the FSC-H signal.
Compensation is set to the values defined by the last Instrument
QC run performed, unless user defined compensation is selected.

To open a new workspace:

1. Do one of the following:

• If the software is not already open, double-click the

BD Accuri C6 Plus software icon (or BD CSampler Plus
software icon) on the desktop. The software opens with a
new workspace.
• If the software is already open, select File > New
workspace. Only one workspace can be open at a time. If
desired, save any unsaved changes to the previous
workspace when prompted.
A new workspace can be used to collect a new data set and create
an analysis template.
 Chapter 9: Managing data 189

Creating a workspace template

Introduction This topic describes the workspace template and provides
instructions for creating a new template.

About templates A workspace template contains a predefined workspace for quick

and easy setup and analysis.

All markers, regions, gates, parameter names, and sample names

are saved without any data points. Acquisition settings are saved
based on the currently selected sample. If you selected User
Defined compensation settings, those settings will also be saved.

Several templates are available at bdbiosciences.com. You can also

create your own customized templates. Templates created in
BD Accuri C6 software are compatible with BD Accuri C6 Plus
software.

Creating a To create a template:

workspace 1. Define the plots, gating, and acquisition settings in a blank
template workspace, or use the current .ci file.

2. Select File > Save template as.

3. Navigate to the location to save the file, enter the file name,
and click Save. The file is saved with the extension .cit.
190 BD Accuri C6 Plus System User’s Guide

Exporting and importing data

Introduction This topic describes how to export or import files.

Exporting data You can export sample data, including compensation settings,
from all or individual sample wells as FCS 3.1 files. No gates or
analysis settings are exported with the data. You can also export
plot data and sample settings.

Note: Exported files are not compatible with BD CellQuest Pro or

BD FACSDiva™ software.

To export data:
1. Do one of the following:

• Select File > Export FCS File to export and save the data
from the currently selected well as an FCS 3.1 file. Enter a
file name and click Save.
• Select File > Export ALL Samples as FCS to export and
save the data for all wells as individual FCS 3.1 files. A
default folder is created on the desktop. Click Ok.

• Select File > Export Plot Data as CSV to save an individual

file in CSV format with the information for each event in
the well. Enter a file name and click Save.
• Select File > Export Sample Settings to export acquisition
criteria, sample names, parameter names, and
compensation values to a CSV file. This option is available
only for BD CSampler Plus software.
 Chapter 9: Managing data 191

Importing data Only FCS files created using BD Accuri C6 Plus software or
BD Accuri C6 software can be imported into a workspace file or
template.

To import an FCS data file into the software:

1. Select an empty data well in a workspace file or template. If
the currently selected data well contains data, the software will
start FCS file import in the first empty data well.

2. Select File > Import FCS File.

3. Navigate to the file(s), select it, and click Open.

Multiple FCS files from within a folder can be selected and

imported as a group using Shift + click. Files begin importing
into the selected data well and proceed in succession,
horizontally, from left to right, wrapping to the following row
once the current row is filled.
192 BD Accuri C6 Plus System User’s Guide
 10
Maintenance
This chapter covers the following topics:

• Maintenance schedule (page 194)

• Cleaning the outside of the instrument (page 196)
• Backflushing the SIP (page 197)
• Performing a SIP clean (page 198)
• Cleaning the fluidics (page 200)
• Running an extended flow cell clean (page 200)
• Cleaning the fluid bottles (page 202)
• Replacing the fluid bottle filters (page 203)
• Replacing the in-line sheath filter (page 204)
• Replacing the peristaltic pump tubing (page 206)
• Collecting precise volume measurements (page 214)
• Purging the fluid sensor lines (page 209)
• Unclogging the SIP (page 211)
• Collecting precise volume measurements (page 214)
• Aligning the CSampler Plus after a collision (page 217)
• Restarting the system after storage (page 219)
194 BD Accuri C6 Plus System User’s Guide

Maintenance schedule
Introduction This topic provides a list of daily, scheduled, and unscheduled
maintenance procedures.

Daily maintenance Daily maintenance is part of the startup and shutdown procedures.
See Starting up the system (page 41) and Shutting down the system
(page 43). Additionally, the following procedures can be
performed as needed.

Task When to perform

Backflushing the SIP (page 197) When you suspect a clog in the SIP

Performing a SIP clean At the completion of every run or

(page 198) more often if desired

Cleaning the fluidics (page 200) Occurs automatically during

shutdown

Scheduled A dialog is displayed every 60 days (or when 35 L of sheath fluid

maintenance has been used) to remind you to perform scheduled maintenance.
When the dialog appears, complete the maintenance, select the
maintenance complete option, and click OK. If you select Remind
me later, the message will appear again in 5 days.
 Chapter 10: Maintenance 195

Scheduled maintenance should be performed as described in the

following table.

Task When to perform

Replacing the fluid bottle filters (page 203) Every 2 months

Replacing the in-line sheath filter Every 2 months

(page 204)

Replacing the peristaltic pump tubing Every 2 months

(page 206)

Cleaning the fluid bottles (page 202) Once a month

Unscheduled Perform the following procedures as needed.

maintenance
Task When to perform
Cleaning the outside of the At the end of each day or as
instrument (page 196) needed

Filling the fluid bottles (page 36) As needed or when prompted by

the software

Emptying the waste bottle As needed or when prompted by

(page 39) the software

Running an extended flow cell When instructed by tech support

clean (page 200) or if you notice a decrease in
instrument sensitivity
196 BD Accuri C6 Plus System User’s Guide

Cleaning the outside of the instrument

Introduction This topic describes how to wipe down the instrument.

Required materials • DI water

• 10% bleach solution, BD FACSClean solution, or 70%
ethanol
• Paper towels or disposable wipes

Procedure To wipe down the outside of the instrument:

1. Moisten a paper towel with the cleaning solution.

2. Wipe down the outside panels and the sample stage. If you
have a CSampler Plus, wipe down the CSampler Plus tray and
mat.

3. Moisten a clean paper towel with DI water and wipe the areas
you wiped with the cleaning solution.

4. Dispose of the paper towels as biohazard waste.

Caution: Biohazard! Dispose of the paper towels as

biohazard waste.
 Chapter 10: Maintenance 197

Backflushing the SIP

Introduction This topic describes how to run the backflush cycle to remove clogs
at the base of the SIP.

About the The backflush cycle forces fluid out of the flow cell and out the SIP
backflush cycle to remove bubbles in the flow cell and/or clogs in the SIP. Perform
a backflush if you notice the event rate slowing or stopping and
you suspect a clog.

Backflushing when To perform a backflush when running manually:

running manually 1. Place an empty tube on the SIP.

2. Select Instrument > Run Backflush cycle.

You can also click Backflush from the control panel of the
Collect or Manual Collect tab.

3. Click Backflush in the dialog prompting you to load an empty

tube.

4. When the backflush is complete, discard the tube in the same

way as you would any biohazardous sample to ensure no
possible safety risk.

Backflushing when To perform a backflush using the CSampler Plus:

using a CSampler 1. Ensure a tube containing 2 mL of DI water is located in the
Plus square location ( ).

2. Select Instrument > Run Backflush cycle.

You can also click Backflush from the control panel of the
Collect or Manual Collect tab.
198 BD Accuri C6 Plus System User’s Guide

Performing a SIP clean

Introduction This topic describes how to clean the SIP with BD FACSClean.

About SIP cleans The SIP clean runs BD FACSClean through the SIP for
approximately 5 minutes followed by water for approximately
5 minutes. Regular SIP cleans are vital for keeping your fluidics
system clear and free of clogs.

When to run a SIP We recommend always running a SIP clean at the end of each run.
clean You can also run a SIP clean as often as you like to prevent SIP
clogs.

Caution: If you leave the instrument idle for 15 minutes

or longer when running samples, run a SIP clean before
resuming operation to clear the SIP and prevent clogs.

Required materials • 2 mL of BD FACSClean

• 2 mL of fresh DI water

Performing a SIP To perform a SIP clean when running manually:

clean when running 1. Click SIP Clean on the control panel of the Collect or Manual
manually Collect tab.

A dialog prompts you to load a tube containing 2 mL of

BD FACSClean.

2. Load the tube of BD FACSClean and click Clean.

When step 1 is complete, a dialog prompts you to load a tube

containing 2 mL of water.

3. Load the tube of water and click Clean.

 Chapter 10: Maintenance 199

Performing a SIP To perform a SIP clean using the CSampler Plus:

clean when using a 1. Click SIP Clean in the control panel of the Collect or Manual
CSampler Plus Collect tab.

A dialog opens prompting you to load tubes of BD FACSClean

and water.

2. Ensure the following tubes are in the designated locations on

the CSampler Plus. If necessary, click Eject Rack and insert the
tubes, then click Load Rack.

• 2 mL of BD FACSClean in the triangle location ( )

• 2 mL of DI water in the circle location ( )
• Also ensure the square ( ) location has a tube containing
2 mL of DI water, as the SIP is left in this tube after a SIP
clean.
3. Click SIP Clean.
200 BD Accuri C6 Plus System User’s Guide

Cleaning the fluidics

Introduction This topic describes how to clean the fluidics lines using the Clean
Fluidics cycle.

About cleaning the The fluidics cleaning cycle is automatically run when you shut
fluidics down the instrument. You can also run it at any time, if necessary.
During the fluidics cleaning cycle, BD FACSClean is run through
the fluid lines, followed by sheath. A second cycle runs detergent
solution through the fluid lines, followed by sheath.

The fluidics cleaning takes about 13 minutes.

Required materials • 2 mL of DI water

Cleaning the To clean the fluidics when running manually:

fluidics when 1. Place a tube containing 2 mL of DI water on the SIP.
running manually
2. Select Instrument > Run Clean Fluidics.

Cleaning the To clean the fluidics when using a CSampler Plus:

fluidics when using 1. Ensure that a tube containing 2 mL of DI water is located in
a CSampler Plus the square location ( )

2. Select Instrument > Run Clean Fluidics.

Running an extended flow cell clean

Introduction This topic describes how to run an extended flow cell clean cycle.

About an extended An extended flow cell clean cycle provides a way to thoroughly
flow cell clean clean the flow cell. Run an extended flow cell clean cycle if you
notice excessive debris or high CVs during instrument QC. During
 Chapter 10: Maintenance 201

extended flow cell cleaning, the flow cell fills completely with
cleaning solution from a sample tube on the SIP. The cycle
automatically shuts down the cytometer, allowing the flow cell to
soak with the cleaning solution.

Required materials • 2 mL of BD Extended Flow Cell Clean Solution

Procedure To run an extended flow cell clean:

1. Place a tube with at least 500 µL of Extended Flow Cell Clean
Solution on the SIP.

Caution! Never run an extended flow cell clean

cycle without a tube containing at least 500 µL of
fluid.
2. Select Instrument > Extended Flow Cell Clean.

A dialog opens prompting you to install a tube of Flow Cell

Cleaner on the SIP if running manually, or load a rack with the
solution in position A01 if using a CSampler Plus.

3. Load the tube of Extended Flow Cell Clean Solution and click
Proceed.

4. After the cytometer is shut down, leave it off for at least

2 hours (or overnight, for a more thorough cleaning).

5. Restart the cytometer.

To thoroughly rinse the flow cell, the cytometer performs a full

fluidics cleaning cycle at startup and the software displays the
message Extra startup time needed due to cleaning or
improper shutdown.

Note: This message also appears after a power outage if the

instrument was forced to shut down.

6. Discard the tube once startup is complete.

202 BD Accuri C6 Plus System User’s Guide

Cleaning the fluid bottles

Introduction This topic describes how to clean the sheath, waste, detergent
solution, and FACSClean bottles.

About cleaning the Clean the bottles every month to ensure that residue and
bottles contaminants do not build up inside the bottles. Do not allow the
bottle filters to dry out while you clean the bottles.

Required materials • 10% bleach solution

• DI water

Procedure To clean the fluid bottles:

1. Disconnect and empty all the bottles.

2. Rinse the bottles with DI water.

3. Rinse the bottles with a 10% bleach solution.

4. Thoroughly rinse the bottles with DI water 2–3 times to

remove the bleach residue.

5. Refill each bottle with the appropriate fluid. See Filling the
fluid bottles (page 36).

6. Replace the caps on the fluid bottles.

7. Snap the color-coded tubing back into the fittings by pushing

firmly until you hear a click.
 Chapter 10: Maintenance 203

Replacing the fluid bottle filters

Introduction This topic describes how to replace the fluid bottle filters.

About fluid bottle The sheath, BD FACSClean, and detergent bottles each contain a
filters disk filter. We recommend replacing these filters every 2 months.

Caution: Biohazard! Contact with biological specimens

and materials can transmit a potentially fatal disease.
Wear suitable protective clothing, eyewear, and gloves.

Required materials • Three fluid filters (one for each bottle)

Procedure To replace the fluidic bottle filters:

1. Disconnect the quick-connect lines from the top of each bottle.

2. Carefully remove the lid from each bottle.

3. Disconnect the filter Luer connector at the end of the fluidic

tubing. Discard the filter as you would a biological sample.

2
204 BD Accuri C6 Plus System User’s Guide

The following table describes the components of the bottle cap

assembly.

No. Component Description

1 Fluid sensing line Detects fluid in the bottle.

2 Disk filter Filters the fluid from the bottle.

4. Replace the filter with the same filter type.

5. Reassemble the bottles and reconnect the quick-connect lines.

Replacing the in-line sheath filter

Introduction This topic describes how to replace the in-line sheath filter.

When to replace We recommend replacing the in-line sheath filter every 2 months.
the in-line sheath
filter If you notice a yellow discoloration, fluid leaking, or if the filter is
less than 50% full, change the filter immediately.

Required materials • In-line sheath filter

Before you begin Write today’s date on the new in-line sheath filter to help you keep
track of unscheduled filter replacements.

Procedure To replace the in-line sheath filter:

1. Turn off the cytometer.

The Clean Fluidics cycle runs for approximately 13 minutes,

then the cytometer powers off automatically.

2. Lift the cytometer lid and remove the plastic storage bin.
 Chapter 10: Maintenance 205

3. Each time you change the in-line sheath filter, visually inspect
all tubing and connectors for fluid leaks.

Look for liquid, dried residue, or discoloration of the metal

surfaces anywhere near the tubing. If you notice any evidence
of a leak, contact BD Biosciences Technical Support.

Caution: Biohazard! Any evidence of fluid coming

from, or near, a red or clear line should be
considered a biological hazard.

Caution: Biohazard! Wear suitable protective

clothing, eyewear, and gloves. Dispose of waste using
proper precautions and in accordance with local
regulations.
4. Twist the Luer locks on both ends of the in-line sheath filter to
disconnect the locks. Do not pull on the tubing.

5. Discard the filter.

Although sample material does not pass through this filter, we

recommend discarding it in the same way as any biohazardous
sample to ensure no possible safety risk.

6. Install a new in-line sheath filter by reconnecting the Luer

fittings.

This filter has a male and female end to ensure that it can only
be installed in the correct orientation.
206 BD Accuri C6 Plus System User’s Guide

7. Replace the plastic storage bin and close the cytometer lid.

8. Place a sample tube with DI water on the SIP, or if using a

CSampler Plus, make sure there is a tube with 2 mL of water in
the square location ( ).

9. Turn on the cytometer.

Note: Upon startup you may see an error message indicating that
a fluidics system error was detected. This error is normal after
replacing the in-line sheath filter. See Hardware fault errors
(page 225) for information.

Replacing the peristaltic pump tubing

Introduction This topic describes how to replace the tubing for the peristaltic
pumps. The cytometer includes two peristaltic pumps—a sheath
pump and a waste pump.

About this Replace the pump tubing every 2 months. We recommend

procedure replacing both pieces of tubing at the same time.

Caution: Moving parts! Turn off the cytometer before

changing the tubing.

Tubing comes in contact with biological samples and therefore

should be considered hazardous.

Caution: Biohazard! Contact with biological specimens

and materials can transmit a potentially fatal disease.
Wear suitable protective clothing, eyewear, and gloves.

Required materials • Peristaltic pump tubing

 Chapter 10: Maintenance 207

Procedure To replace the pump tubing:

1. Turn off the cytometer.

The Clean Fluidics cycle runs for approximately 13 minutes,

then the cytometer powers off automatically.

2. Lift the cytometer lid.

3. Squeeze the grip marks on the pump retainer clip to remove

the clip.

4. Carefully pull the Luer connectors outward, sliding the fittings

off the pump head.
208 BD Accuri C6 Plus System User’s Guide

5. Disconnect the tubing by unscrewing the plastic Luer

connectors (a total of four connectors—two for each pump).

Blue tubing is connected to the sheath pump and red tubing to

the waste pump.

6. Remove the pump tubing from the pump head and discard it
as you would a biological sample according to standard
laboratory protocols and regulations.

Sample passes through this pump tubing. Consider it

biologically hazardous.

7. Install new peristaltic pump tubing by sliding the Luer fittings

on the pump head and snapping the fitting in place.

8. Replace the pump retainer clip.

9. Reconnect the tubing to the Luer connectors.

Caution! Ensure that the Luer fittings are connected

to the correct tubing elements. Also ensure that when
you tighten the fittings, the tubing does not twist or
kink. If it does twist, unscrew the Luer fitting, turn it
counter-clockwise, then retighten it.
10. Gently close the cytometer lid.

11. Place a sample tube with DI water on the SIP, or if using a

CSampler Plus, make sure there is a tube with 2 mL of water in
the square location ( ).

12. Turn on the cytometer.

 Chapter 10: Maintenance 209

Note: Upon startup you may see an error message indicating that
a fluidics system error was detected. This error is normal after
replacing the peristaltic pump tubing. See Hardware fault errors
(page 225) for information.

Purging the fluid sensor lines

Introduction This topic describes how to purge the fluid sensing lines. See
Replacing the fluid bottle filters (page 203) for information on the
fluid sensing lines.

About this Perform this procedure if the traffic light message indicates that the
procedure sheath is empty or the waste is full, when in fact all fluid levels are
as they should be. Fluid in the sensing lines can lead to erroneous
tank level messages.

Before you begin Start with the instrument turned off. Ensure the sheath bottle is full
and the waste bottle is empty, except for bleach. Place the sheath
bottle on the benchtop where you can monitor the fluid level.

Procedure 1. Lift the cytometer lid and remove the plastic storage bin.

2. Locate the round, finger-size access hole near the in-line sheath
filter.
210 BD Accuri C6 Plus System User’s Guide

3. Place your finger over the pin hole inside the access hole.

4. With your finger sealing the pin hole, turn on the cytometer.

5. Keep your finger over the hole for 30 seconds. You should see
continuous bubbling in the sheath bottle.

6. After 30 seconds, remove your finger.

7. Replace the storage bin and close the lid to allow the
cytometer to complete the normal fluidics startup.
 Chapter 10: Maintenance 211

Unclogging the SIP

Introduction This topic describes how to use a syringe to unclog and rinse the
SIP.

About this Perform the syringe SIP unclog if the SIP is clogged and performing
procedure backflushes and SIP cleans have not fixed the problem.

Required materials • Two 3-mL syringe tools (assembled)

• BD FACSClean
• DI water

Before you begin Before using the syringe to unclog the SIP, try unclogging the SIP
by performing several backflushes followed by several SIP cleans.

Assembling the Assemble two syringe tools as follows:

syringes
1. Connect the female Luer connector, tubing, and quick-connect
coupling.

2. Connect the tubing assembly to a 3-mL syringe, as shown.

Unclogging the SIP To unclog the SIP:

1. Turn off the cytometer.

The Clean Fluidics cycle runs for approximately 13 minutes,

then the cytometer powers off automatically.

2. Lift the cytometer lid.

212 BD Accuri C6 Plus System User’s Guide

3. Disconnect the three flow cell fluid lines (blue sheath, red
waste, clear purge) from the chassis by turning each connector
approximately 1/8 turn counter-clockwise.

The waste and purge lines come in contact with biological

samples and therefore should be considered hazardous.

Caution! Contact with biological specimens and

materials can transmit a potentially fatal disease.
Wear suitable protective clothing, eyewear, and
gloves.
4. Connect the blue (sheath) and red (waste) lines to each other
by aligning the connectors together and twisting slightly.

5. Fill the first syringe with BD FACSClean.

6. Ensure an empty tube is installed on the SIP.

 Chapter 10: Maintenance 213

7. Connect the syringe to the clear (purge) line by aligning the

connectors and twisting slightly.

8. Slowly depress the plunger on the syringe, pushing the cleaning

solution into the flow cell and out the SIP.

9. Slowly pull the plunger to pull the cleaning solution back into
the syringe.

10. Repeat the push/pull cycle a few more times to ensure the clog
is cleared. Finish by pushing the BD FACSClean into the tube.

Rinsing the SIP To rinse the SIP:

1. Fill the second syringe with DI water.

2. Install an empty tube on the SIP.

Note: If performing this procedure with a CSampler Plus,

ensure that the tube in the quare ( ) location, in which the
SIP is parked, does not overflow.

3. Slowly depress the plunger on the syringe, pushing water into

the flow cell and out the SIP.

4. Slowly pull the plunger to pull the water back into the syringe.
214 BD Accuri C6 Plus System User’s Guide

5. Repeat the push/pull cycle a few more times to ensure the SIP
is thoroughly rinsed. Finish by pushing the water into the tube.

6. Disconnect the syringe from the clear (purge) line.

7. Disconnect the blue (sheath) and red (waste) lines from each
other.

8. Reconnect the three lines to the connectors in the chassis by

turning each connector approximately 1/8 turn clockwise until
they click into place.

Collecting precise volume measurements

Introduction This topic describes how to use the BD Accuri C6 Plus flow
cytometer for precise volume measurements.

About precise Each BD Accuri C6 Plus flow cytometer is calibrated at the factory
volume for accurate volume measurement. This enables the system to
measurements provide absolute cell counts per sample volume. The system
displays the volume in µL and automatically calculates counts per
µL for any gated population. Follow this procedure when accurate
volume measurement is required during sample acquisition. For
more detailed information, refer to the Technical Bulletin, A Guide
to Absolute Cell Counting on the BD Accuri C6 Flow Cytometer,
which can be found at bdbiosciences.com.

Before you begin Ensure that the peristaltic pump tubing and in-line sheath filter
have been replaced within the last 60 days.

General guidelines Follow these general guidelines:

• Use 12 x 75-mm tubes (any type of plastic).

• Use Medium or Fast flow rate to measure direct volume. Do
not use the Slow flow rate.
 Chapter 10: Maintenance 215

• Use sample volumes between 300 µL and 2 mL. Never acquire

more than 750 µL from a single tube on the Medium fluidics
setting or 1,500 µL on the Fast fluidics setting.
• Adjust sample concentrations to fall approximately within the
range of 1 x 103 and 5 x 106 cells or particles/mL.
• Acquire data on the Medium or Fast fluidics setting only.
Custom fluidics settings above 15 µL and 16 µm may be used,
but must be validated by an independent control/count bead.
• Only acquire once from any sample tube. Sample height within
the tube is critical. Replicate measurements must be obtained
from separate sample tubes made by aliquoting sample into
the appropriate number of equal volumes.
• Always compare the same stop limit types. Do not compare
concentrations collected with volume stop counts to those
collected with event stop counts.
• Always validate accurate counting by using a reference count
bead in the experimental buffer, using the same sample volume
and tube as in the experiment. If bead counts are within 20%
of the expected value (based on information provided by the
bead manufacturer), proceed with sample collection. If bead
counts are not within 20% of expected values, proceed with
auto-adjusting the volume settings as described below.

Auto-adjust the To auto-adjust the volume settings:

volume settings 1. Ensure that the fluid levels in the sheath, BD FACSClean, and
detergent solution bottles are filled and that there are no
kinked fluidic lines.

2. Restart the cytometer to flush the fluid lines. When startup is

complete the traffic light turns green and the software displays
the message Cytometer is connected and ready.

3. Within 5 minutes of the completion of the startup cycle, place

a 12 x 75-mm tube containing 750 µL of 70% ethanol on the
SIP. Acquire 400 µL on the Fast fluidics setting.

4. Within 5 minutes of completion of the ethanol run, place a

12 x 75-mm tube containing 1,500 µL of filtered DI water on
the SIP. Acquire 400 µL on the Fast fluidics setting.
216 BD Accuri C6 Plus System User’s Guide

5. Within 5 minutes of completion of the water run, place a

sample on the SIP. See the following bullets for sample
requirements. Select Instrument > Auto-adjust volume settings.

• The volume setting adjustment should be performed in the

same tube as the experimental sample.
• The volume setting adjustment should be performed using
a sample of the same or similar viscosity as the samples to
be analyzed. For example, if lysed human peripheral blood
samples are to be acquired, then lysed human peripheral
blood should be used during the volume setting adjustment.
• The volume in the control sample tube should be 110 µL
more than the volume used with subsequent test samples.
For example, if using 1,000-µL samples, perform auto-
adjust volume settings with 1,110 µL in the tube. The
adjustment procedure consumes approximately 220 µL.
The values determined by the cytometer are based on the
average sample height in the tube during the auto-adjust
volume procedure.
• If sample volumes >50 µL are to be acquired from the
sample tube, the auto-adjust volume should take this into
account and the average volume in the sample tube during
the acquisition should be used. For example, if 100 µL are
to be acquired from a 1,000-µL sample, the auto-adjust
volume would be 950 µL. The average volume [(Starting
Volume + Ending Volume)/2] during the acquisition is
[(1,000+900)/2] or 950 µL.
6. The cytometer will perform an auto-adjust volume cycle,
taking approximately 13 minutes. Once completed, the traffic
light will revert to green and display the Cytometer is
connected and ready message. Repeat fluidics validation using
a reference count bead as described above.

7. If the status message indicates the auto-adjust volume

procedure was successful, repeat the performance validation. If
the status message indicates that procedure failed, see Auto-
adjust volume settings procedure failed (page 231).

The cytometer will operate normally after a failed auto-adjust

volume procedure. However, volume measurements for the
 Chapter 10: Maintenance 217

samples might be incorrect, since the cytometer reverts to the

factory-set default fluidics volume settings. All other aspects of
the data will be normal.

Aligning the CSampler Plus after a collision

Introduction This topic describes how to perform a manual alignment of the
rack to the SIP if the CSampler Plus arm collides with an object in
its path.

About alignment The CSampler Plus performs an alignment to verify that the tube
rack is aligned to the SIP every time the flow cytometer is powered
up or if the CSampler Plus arm collides into an object. A manual
alignment can be performed using the software at any time.

If there is an obstruction in the path of the CSampler Plus, the

software displays a red traffic light and opens a dialog indicating
that a collision has occurred.

Procedure To perform an alignment:

1. Remove any objects from the CSampler Plus mat.

2. Do one of the following:

• If a collision has occurred, click Align in the Collision

Detected dialog.
• If a collision did not occur, select Instrument > Align
CSampler Plus.
218 BD Accuri C6 Plus System User’s Guide

If a second collision occurs, the software automatically performs a

second alignment. If the second alignment fails, contact
BD Biosciences Technical Support.

Samples left in the tube rack can be recovered by turning off the
cytometer and gently pushing down on the white cylindrical motor
housing.
 Chapter 10: Maintenance 219

Restarting the system after storage

Introduction This topic explains how to prepare the BD Accuri C6 Plus flow
cytometer for use after it has been stored for a period of time.

Procedure 1. Replace all fluid bottle filters—sheath, BD FACSClean, and

detergent. See Replacing the fluid bottle filters (page 203).

2. Replace the in-line sheath filter. See Replacing the in-line

sheath filter (page 204).

3. Fill the fluid bottles with fresh fluid and ensure the waste
bottle is empty except for 200 mL of bleach. See Filling the
fluid bottles (page 36).

4. Turn on the cytometer. See Starting up the system (page 41).

Allow the startup fluid cycle to complete. It takes about
15 minutes.

5. Turn off the cytometer. See Shutting down the system

(page 43). Allow the shutdown fluid cycle to complete. It takes
about 13 minutes.

6. Turn on and off the cytometer two more times, allowing the
fluid cycles to complete before cycling the power.
220 BD Accuri C6 Plus System User’s Guide
 11
Troubleshooting
This chapter covers the following topics:

• Troubleshooting overview (page 222)

• Hardware troubleshooting (page 222)
• Software troubleshooting (page 227)
• Acquisition troubleshooting (page 229)
• QC troubleshooting (page 231)
222 BD Accuri C6 Plus System User’s Guide

Troubleshooting overview
Introduction This topic describes troubleshooting for the BD Accuri C6 Plus
system.

The Troubleshooting chapter lists problems you may encounter

during normal operation. It includes:

• Hardware troubleshooting (page 222)

• Software troubleshooting (page 227)
• Acquisition troubleshooting (page 229)
• QC troubleshooting (page 231)

Additional help If, after reading through the possible problems and recommended
solutions, you still have questions or are experiencing problems,
contact BD Technical Support. See Technical support (page 11) for
information.

Hardware troubleshooting
Introduction This topic describes how to troubleshoot hardware problems with
the cytometer or CSampler Plus.

Cytometer and/or
computer does not Possible causes Recommended solutions
power on
Power supply not Make sure the power supplies and cords are
plugged in plugged into an appropriate outlet.

Power outlet Check the outlet to make sure it is functioning

malfunction properly.
 Chapter 11: Troubleshooting 223

Power and event Power and event indicator lights flash simultaneously
indicator lights
flash on startup Possible causes Recommended solutions
Waste tank full Turn off the power to the cytometer. Empty the
waste bottle, then restart the cytometer.

Power and event indicator lights flash alternately

Possible causes Recommended solutions

CSampler Plus Turn off the power to the cytometer. Check for
failed to align obstructions, then restart the cytometer.
properly

Message “Extra
startup time Possible causes Recommended solutions
needed due to
Extended flow cell Allow the extended startup to run. It will take
cleaning or clean cycle was run. approximately 25 minutes.
improper
shutdown” Fluidics error
occurred during
shutdown.
Instrument forcibly
shut down, for
example power
outage.

USB port not active

or lost connection Possible causes Recommended solutions
Using a USB port on Do not use a USB port on the same hub as the
the same hub as the cytometer. For example, use USB ports on the
cytometer front of the computer for flash drives.

If the USB port is not on the same hub as the

cytometer, unplug the drive, wait 5 seconds,
then plug it back in. If that doesn’t work,
restart the computer.
224 BD Accuri C6 Plus System User’s Guide

CSampler Plus
collision Possible causes Recommended solutions
Collision message Time-out occurred because the CSampler Plus
appears but no did not reach its designation in the time
collision occurred permitted. Select Instrument > Align CSampler
Plus. See Aligning the CSampler Plus after a
collision (page 217)

Fluid leak under

storage bin Possible causes Recommended solutions
In-line sheath filter Check the Luer locks on both ends of the filter
leaking to ensure they are tight. If the filter appears
yellow or is less than 50% filled with fluid,
change it immediately. The filter should be
submerged in fluid.

Peristaltic pump Check the peristaltic pump tubing on both

tubing not properly pumps to ensure it is properly connected to the
installed Luer connectors.

Traffic light
displays message Possible causes Recommended solutions
that waste is full or
Fluid bottle sensing Purge the fluid bottle sensing lines. See Purging
sheath is empty lines have fluid in the fluid sensor lines (page 209).
when they are not them

Message “Your
print job failed” Possible causes Recommended solutions
Printer not Ensure a printer is connected, either locally or
connected or not through a network, and turned on.
turned on

Network down If the printer is connected to a network, ensure

that the network is up and running.
 Chapter 11: Troubleshooting 225

Hardware fault If any of the following errors occur, click Close this window, then
errors follow the steps listed.

Message Recommended solutions

The cytometer 1. Check the fluid levels in sheath, detergent
stopped because a solution, and BD FACSClean bottles.
Fluidic Stability Error 2. Check the retainer clips on the fluid pumps
occurred. to ensure they are properly installed.
3. Perform a backflush, followed by a SIP
clean.
4. Check the system’s performance by
running instrument QC.
5. If you are running a sample, check the
sample tube to ensure adequate sample,
then rerun the sample.
6. If the problem persists, contact
BD Technical Support.

The cytometer 1. Check the system’s performance by

stopped because a running instrument QC.
Red/Blue Laser Error 2. Rerun the sample.
occurred.
3. If the problem persists, contact
BD Technical Support.

The cytometer 1. Rerun the sample.

stopped because a 2. If the problem persists, contact
Signal Processing BD Technical Support.
Error occurred.
226 BD Accuri C6 Plus System User’s Guide

Message Recommended solutions

The cytometer Contact BD Technical Support.
stopped because an
Electronic System
Error occurred.

A Fluidics System If the in-line sheath filter or peristaltic pump

Error was detected. tubing was changed, this message is normal.
1. Perform two to three backflush cycles.
2. Turn off the cytometer, then restart it.
3. If the error appears again, restart the
cytometer a second time.
4. If the problem persists after two shutdown
and startup cycles, contact BD Technical
Support.

If the in-line sheath filter was not just changed:

1. Check the fluid bottles—fill the sheath and
cleaning bottles and/or empty the waste.
2. Check the BD FACSClean (circle) and DI
water (triangle) tubes on the CSampler Plus
tray, if using a CSampler Plus, to ensure
they contain fluid.
3. If running samples, ensure there is fluid in
the sample tube.
4. Check the connections to the fluid bottles,
filter, and pumps.
5. Turn off the cytometer, then restart it.
6. If the problem persists, contact
BD Technical Support.
 Chapter 11: Troubleshooting 227

Software troubleshooting
Introduction This topic describes problems related to the software.

Software hangs at
startup and will not Possible causes Recommended solutions
launch
Software launching Software requires more time to launch. Give
slowly the application time to launch.

USB flash drive Do not use a USB port on the same hub as the
plugged into same cytometer. For example, use USB ports on the
hub as cytometer, front of the computer for external flash drives.
disrupting
 Disconnect flash drive.
communication
 Disconnect the USB cable between the
cytometer and computer, then reconnect it.

Javaw.exe process Use the task manager to end the javaw.exe

interfering process.
1. Press Ctrl + Alt + Delete.
2. Click Start Task Manager.
3. Click the Processes tab.
4. Select javaw.exe and click End Process.
If the process still won’t quit, unplug the USB
cable from the cytometer, then plug it back in.

The traffic light

does not turn green Possible causes Recommended solutions
USB cable not 1. Make sure the USB cable is properly
properly connecting connected between the cytometer and
the cytometer to the computer. It may take several seconds for
computer the port to recognize the cytometer.
2. If necessary, restart the computer.

Switch the USB cable to a different port on the

computer.
228 BD Accuri C6 Plus System User’s Guide

Software not
responding Possible causes Recommended solutions
USB flash drive Do not use a USB port on the same hub as the
plugged into same cytometer. For example, use USB ports on the
hub as cytometer, front of the computer for external flash drives.
disrupting
 Disconnect the flash drive.
communication
 Disconnect either end of the USB cable
between the cytometer and computer, then
reconnect it.

Javaw.exe process Use the task manager to end the javaw.exe

interfering process.
1. Press Ctrl + Alt + Delete.
2. Click Start Task Manager.
3. Click the Processes tab.
4. Select javaw.exe and click End Process.
If the process still won’t quit, unplug the USB
cable from the cytometer, then plug it back in.

USB cable Ensure the USB cable between the cytometer

disconnected and the computer workstation is connected.

Software appears
to shut down but is Possible causes Recommended solutions
still running in the
Javaw.exe process Use the task manager to end the javaw.exe
background interfering process.
1. Press Ctrl + Alt + Delete.
2. Click Start Task Manager.
3. Click the Processes tab.
4. Select javaw.exe and click End Process.
If the process still won’t quit, unplug the USB
cable from the cytometer, then plug it back in.
 Chapter 11: Troubleshooting 229

Acquisition troubleshooting
Introduction This topic describes how to troubleshoot problems you encounter
during acquisition, including the data displayed in the plots.

Data does not look

as expected Possible causes Recommended solutions
Sample needs to be Abort and restart run. Or, rerun sample at
restarted completion of run.

Fluidics dirty or 1. Perform a backflush.

clogged 2. Perform a SIP clean.

Air bubbles in flow 1. Perform a backflush.

cell or in-line sheath 2. Perform a SIP clean.
filter

Event rate drops

during acquisition Possible causes Recommended solutions
Cells settled in Pause acquisition and mix the tube. If using a
bottom of tube CSampler Plus, eject the rack and manually mix
the tubes.

Fluidics dirty or 1. Perform a backflush.

clogged 2. Perform a SIP clean.
3. If the problem persists, perform an
extended flow cell clean cycle.
230 BD Accuri C6 Plus System User’s Guide

Pumps are running

normally, but data Possible causes Recommended solutions
is not being
Sheath too low and/ Check the levels in the sheath and waste
acquired or waste too high bottles.

Fluidics lines Check all fluidics lines, at bottles, harness, and

crimped peristaltic pumps, to see if any are crimped.

SIP clogged 1. Perform a backflush.

2. Perform a SIP clean.
3. If the problem persists, contact
BD Technical Support.

Peristaltic pump Make sure peristaltic pump tubing and retainer

tubing not attached clip are properly attached.
properly

Pumps are running

continually Possible causes Recommended solutions
Air in the in-line  If the filter is less than 50% filled with
sheath filter fluid, replace it.
 If the filter contains an air pocket, perform
a backflush, followed by a SIP clean.

Fluidics lines 1. Perform a backflush.

clogged 2. Perform a SIP clean.

Peristaltic pump Replace peristaltic pump tubing.

tubing damaged
 Chapter 11: Troubleshooting 231

Auto-adjust volume
settings procedure Possible causes Recommended solutions
failed
System fluidics not Perform a Clean Fluidics cycle, then repeat the
clean procedure.

Sample type or Repeat the procedure with a new sample. Make

volume incorrect sure the tube did not run dry during the
procedure.

Peristaltic pump If after performing the solutions above, the

tubing needs procedure continues to fail, replace the
replaced peristaltic pump tubing and repeat the
procedure.

QC troubleshooting
Introduction This topic describes how to troubleshoot problems with
instrument QC.

No beads were
detected Possible causes Recommended solutions
No beads in sample Make sure you are running the correct sample
tube.

Beads not properly  Vortex bead vial before preparing bead

mixed suspension.
 Vortex bead suspension before running
beads.
232 BD Accuri C6 Plus System User’s Guide

QC result failed
Possible causes Recommended solutions
Regions and/or Adjust regions and markers, as necessary. See
markers not set Adjusting the bead regions and markers
correctly (page 53).

Expired beads Check bead expiration date. If necessary, rerun

QC with new beads.

Old bead Once prepared, beads are stable for 8 hours if

suspension stored at 2–8°C and protected from light. After
this period, prepare a fresh bead suspension.

High CVs caused by 1. Perform a backflush.

bubbles 2. Perform a SIP clean.

QC messages
Message Recommended solutions
Event count for Mid  Beads settled in bottom of tube. Resuspend
+ Bright too low the beads and rerun the sample.
 Ensure that the region is encompassing the
mid + bright bead population in the FSC vs
SSC plot. Adjust the region, if necessary.

Percentage of events  Beads settled in bottom of tube. Resuspend

for Mid + Bright too the beads and rerun the sample.
low  Ensure that the region is encompassing the
mid + bright bead population in the FSC vs
SSC plot. Adjust the region, if necessary.
 Beads are no longer stable. Prepare a fresh
bead solution.
 Chapter 11: Troubleshooting 233

Message Recommended solutions

Percentage of events Rerun the beads. If the message appears again,
in FSC/SSC/FL1/FL2/ prepare a new bead suspension. Do not adjust
FL3/FL4 Noise too the noise gates. If the problem persists, contact
low BD Technical Support.

Event count for FL1/ Make sure the bead peak in the corresponding
FL2/FL3/FL4 Bright plot is located within the markers. Adjust the
too low marker, if necessary. If the message still
appears, prepare a new bead suspension.

Percentage of events Make sure the bead peak in the corresponding

in FL1/FL2/FL3/FL4 plot is located within the markers. Adjust the
Bright too low marker, if necessary. If the message still
appears, prepare a new bead suspension.

Bright bead median As seen in the Levey-Jennings plots.

and/or %rCV drift
over time
Possible causes Recommended solutions
Peristaltic pump Change the peristaltic pump tubing.
tubing worn
234 BD Accuri C6 Plus System User’s Guide
 12
Selectable Lasers Module
This chapter covers the following topics:

• Selectable lasers (page 236)

• Annotating selected laser configuration (page 238)
• Optical filter placement (page 240)
• Selectable laser application examples (page 243)
236 BD Accuri C6 Plus System User’s Guide

Selectable lasers
Introduction This topic describes the Selectable Lasers Module option and how
to install, validate, and configure the lasers and optical filters.

The Selectable Lasers upgrade requires prior installation of C6 Plus

software or CSampler Plus software.

About selectable The detectors and lasers of the BD Accuri C6 Plus flow cytometer
lasers operate in a predefined standard configuration: FL1, FL2, and FL3
detect blue laser-excited fluorescence emissions and FL4 detects red
laser-excited emissions. This configuration is referred to as 3 blue 1
red.

Configuration Detector Laser emissions

3 blue 1 red FL1, FL2, and FL3 blue laser-excited emissions

FL4 red laser-excited emissions

The Selectable Lasers Module allows you to operate the cytometer

in two other configurations to significantly expand the
fluorochrome combinations that can be analyzed. See Optical filter
placement (page 240) for examples.

The two configurations include:

Configuration Detector Laser emissions

2 blue 2 red FL1 and FL2 blue laser-excited emissions

FL3 and FL4 red laser-excited emissions

4 blue FL1, FL2, FL3, FL4 blue-laser emissions

Components The standard optical filters supplied with the instrument are 533/
supplied 30 nm (FL1), 585/40 nm (FL2), 670 LP nm (FL3), and 675/25 nm
(FL4).
 Chapter 12: Selectable Lasers Module 237

The Selectable Lasers option comes with the following:

• Selectable Lasers Activation Key (software)

• Three optical filters:
– 780/60
– 610/20
– 630/30

Before you begin • Perform instrument QC and ensure that it passes.

Note: For Instrument QC the laser configuration will
automatically switch to 3 blue 1 red. Ensure that the standard
optical filters are installed when running Instrument QC.
When instrument QC is complete and you close the QC
module, the system will switch back to the configuration that
was previously selected, if different from 3 blue 1 red.

Installing the To install the Selectable Lasers Module:

Selectable Lasers 1. Verify that C6 Plus software or CSampler Plus software is
Module loaded on the computer.

2. Copy the Selectable Lasers Activation Key from the USB drive
to the desktop.

3. Double-click on the Selectable Lasers Activation Key icon.

4. In the installation wizard, choose the correct directory to

install the Activation Key.

The location depends on where C6 Plus software or CSampler

Plus software has been installed. In most cases, the location
will be in one of the following places:

• C:\Program Files\BD Accuri\BD Accuri C6

Plus\ActivationKeys
• C:\Program Files\BD Accuri\BD CSampler
Plus\ActivationKeys
If you have both C6 Plus software and CSampler Plus software
on the same computer, install the Selectable Lasers Activation
Key twice: once in the C6 Plus software directory, and again in
238 BD Accuri C6 Plus System User’s Guide

the CSampler Plus software directory.

5. Click Browse to navigate to the correct location.

6. Click Install.

7. After installation, click Close.

8. Start C6 Plus software or CSampler Plus software. The

Selectable Lasers controls are displayed next to the traffic light
message.

9. Depending on the laser configuration you are going to use, you

may need to replace the standard optical filters with one or
more of the filters that are supplied with the Selectable Lasers
Module.

More information • Annotating selected laser configuration (page 238)

• Optical filter placement (page 240)
• Selectable laser application examples (page 243)

Annotating selected laser configuration

Introduction This topic describes how to properly identify the data wells to
indicate the laser configuration you are using.

About the selected The selectable laser buttons show the most recently selected
laser configuration configuration only. They do not change if you select another data
well with a different configuration. Therefore, we recommend
annotating your data wells in the sample naming field to indicate
the laser configuration used during data collection of each well,
especially when not using the default 3 blue 1 red option. (For
 Chapter 12: Selectable Lasers Module 239

example, name a sample “HPB 4b” to indicate that the 4 blue

option was selected during data collection.)

Laser configurations are not saved in the C6 Plus data file or with a
C6 Plus template. Any previously saved C6 Plus file will default
back to the 3 blue 1 red option when it is opened.

The last laser configuration used to collect a given well of data is

written in the FCS file header using the custom keyword
#LASERCONFIGURATION. FCS file headers can be viewed by
opening an exported FCS file in a text editor (such as Microsoft
Notepad).

More information • Selectable lasers (page 236)

• Optical filter placement (page 240)
• Selectable laser application examples (page 243)
240 BD Accuri C6 Plus System User’s Guide

Optical filter placement

Introduction This topic describes the different optical filter configurations.

About filter Due to the unique optical layout of the cytometer, it is critical that
placement any optional filters used with the Selectable Lasers Module are
placed in the proper positions for optimal performance.

Always use the standard configuration (3 blue 1 red) when running

instrument QC.

Caution! The 630/30 bandpass filter provided with the

Selectable Lasers Kit should only be used when
operating in the 4 blue configuration. Using the 630/30
filter when operating in any other configuration may
damage the corresponding detector due to unfiltered red
laser signal.

Configurations Use the tables below as a guide to optical filter placement for
various fluorochrome combinations.

3 blue 1 red: Configuration 1 (standard filters)

Detector Filter Fluorochrome

FL1 533/30 FITC, GFP, CFSE

FL2 585/40 PE, PI

FL3 670 LP PerCP-Cy5.5, PE-Cy™5, PE-Cy7

FL4 675/25 APC, Alexa-647

3 blue 1 red: Configuration 2

Detector Filter Fluorochrome

FL1 533/30 FITC, GFP, CFSE
 Chapter 12: Selectable Lasers Module 241

Detector Filter Fluorochrome

FL2 585/40 PE

FL3 610/20 PI, PE-Texas Red

FL4 675/25 APC, Alexa-647

When operating in the 3 blue 1 red configuration, place either the

610/20 bandpass or the 670 LP filter in the FL3 position. For best
results when analyzing PE and PE-Texas Red® (PE-TR)
simultaneously, select the filters with the following signal-intensity
considerations in mind:

• PE – bright, PE-TR-moderate to bright: FL3 = 670 LP

• PE – dim to moderate, PE-TR any level: FL3 = 610/20
• PE – bright, PE-TR dim: may be difficult to separate; consider
using the 4 blue configuration with a 630/30 in FL4 to detect
PE-TR.

2 blue 2 red Configuration

Detector Filter Fluorochrome

FL1 533/30 FITC, GFP, CFSE

FL2 585/40 PE, PI, PE-Texas Red

FL3 780/60 APC-Cy7

FL4 675/25 APC, Alexa-647

4 blue: Configuration 1

Detector Filter Fluorochrome

FL1 533/30 FITC, GFP, CFSE

FL2 585/40 PE, PI, PE-Texas Red

FL3 780/60 PE-Cy7

FL4 675/25 PerCP-Cy5.5, PE-Cy5

242 BD Accuri C6 Plus System User’s Guide

4 blue: Configuration 2

Detector Filter Fluorochrome

FL1 533/30 FITC, GFP, CFSE

FL2 585/40 PE

FL3 780/60 PE-Cy7

FL4 610/20 PI

630/30 PE-Texas Red

4 blue: Configuration 3

Detector Filter Fluorochrome

FL1 533/30 FITC, GFP, CFSE

FL2 585/40 PE

FL3 675/25 PerCP-Cy5.5, PE-Cy5

FL4 610/20 PI

630/30 PE-Texas Red

More information • Selectable lasers (page 236)

• Annotating selected laser configuration (page 238)
• Selectable laser application examples (page 243)
 Chapter 12: Selectable Lasers Module 243

Selectable laser application examples

Introduction This topic provides examples of different selectable laser
configurations.

Note: The following examples are from data collected on a

BD Accuri™ C6 flow cytometer.

2 blue 2 red Configuration

configuration
examples Detector Filter Fluorochrome
FL1 533/30 FITC

FL2 585/40 PE

FL3 780/60 APC-Cy7

FL4 675/25 APC

Example 1: 2 blue 2 red

FITC, PE, APC and APC-Cy7: Human Peripheral Blood Subsets

The following images show the results for a 4-color analysis of

CD3+CD4+ cells. Human peripheral blood was stained with
CD3-APC-Cy7, CD4-APC, CD45RA-FITC, and CD45RO-PE.
244 BD Accuri C6 Plus System User’s Guide
 Chapter 12: Selectable Lasers Module 245

Example 2: 2 blue 2 red

BD™ CBA Flex Set

The following images show the results from a BD Cytometric Bead

Array (CBA) 30 Plex bead mixture.

unzoomed, area zoomed, height

4 blue Example 1: 4 blue

configuration FITC, PE, PE-Cy5, PE-Cy7: Human Peripheral Blood Subsets
examples

Detector Filter Fluorochrome

FL1 533/30 FITC

FL2 585/40 PE

FL3 780/60 PE-Cy7

FL4 675/25 PE-Cy5

The following images show CD45-FITC vs SSC gating of

lymphocytes. Human peripheral blood was stained with CD45-
FITC, CD8-PE, CD3-PE-Cy5, and CD4-PE-Cy7.
246 BD Accuri C6 Plus System User’s Guide

unzoomed, area zoomed, area

Example 2: 4 blue
FITC, PE, PE-Cy5, PE-Texas Red: Human Peripheral Blood
Subsets

Detector Filter Fluorochrome

FL1 533/30 FITC

FL2 585/40 PE

FL3 675/25 PE-Cy5

FL4 630/30 PE-Texas Red

The following images show the results of human peripheral blood

stained with CD45-FITC, CD4-PE, CD3-PE-Cy5, and CD8-PE-
TexasRed. The color compensation value to correct the spillover of
PE (FL2) into the PE-Texas Red (FL4) detector may be in the range
of 70% to 90%.
 Chapter 12: Selectable Lasers Module 247

More information • Selectable lasers (page 236)

• Annotating selected laser configuration (page 238)
• Optical filter placement (page 240)
248 BD Accuri C6 Plus System User’s Guide
 13
User Tracking Module
This chapter covers the following topics:

• Tracking user activity (page 250)

• Managing user accounts (page 253)
• Monitoring user activity (page 256)
250 BD Accuri C6 Plus System User’s Guide

Tracking user activity

Introduction This topic describes how to install and start the User Tracking
option.

About user tracking User tracking is an optional upgrade that allows laboratory
administrators to track the activities of cytometer operators by
assigning a user name and password to each individual. The user
name and password are required to log into the software. The
passwords are unrelated to any Windows passwords used on the
host computer or network. You must be the administrator to use
the user tracking features.

Installing the User To install user tracking on your computer:

Tracking module 1. Copy the user tracking activation key (User Tracking
Installer.exe) from the USB drive to the computer desktop.

2. Double-click the User Tracking Installer icon on the desktop.

3. In the installation wizard, select the correct directory to install

the activation key.

The location depends on where the C6 Plus software was

installed. In most cases, the location is in one of two places:

• C:\Program Files\BD Accuri\BD Accuri C6 Plus

\ActivationKeys
• C:\Program Files\BD Accuri\BD CSampler
Plus\ActivationKeys
 Chapter 13: User Tracking Module 251

If necessary, use the browse button to navigate to the correct

folder.

4. Click Install.

The software displays a confirmation message after successful

installation.
252 BD Accuri C6 Plus System User’s Guide

Signing in to the Once the tracking software is installed, users must now sign in
software each time they want to use the C6 Plus software. To use the
tracking feature, sign in to the software as the administrator.

To sign in to the software:

1. Launch the software.

2. When the Sign In dialog is displayed, enter the user name and
password provided to you during installation.

3. Click OK.

4. Use the software as usual.

5. To access the feature, select About > Users.

Signing out of the Be sure you sign out of the software when you are finished using it.
software If you do not sign out, software will continue to log your time.

To sign out of the software:

1. Select File > Quit.

More information • Managing user accounts (page 253)

• Monitoring user activity (page 256)
 Chapter 13: User Tracking Module 253

Managing user accounts

Introduction This topic describes how the administrator can add new users,
delete existing users, and change the administrator password.

Adding user The Users menu option is only visible to the administrator, and
accounts only after the User Tracking software has been installed.

To add new user accounts:

1. Sign in as the administrator.

2. Select About > Users.

3. In the Users dialog, click Add New User.

4. Type a user name and password for the user in the blank fields.

5. Type any notes you want to add in the Notes field.

254 BD Accuri C6 Plus System User’s Guide

This information is only visible in the Users dialog and the

userUsage log. See Monitoring user activity (page 256).

6. Click Save.

Deleting user You cannot delete the administrator account.

accounts
To delete a user account:
1. Select About > Users.

2. Click Delete next to the user account you want to remove.

3. Click Save.
 Chapter 13: User Tracking Module 255

Changing a The administrator can change their password or any user password
password at any time.

Note: If you are the administrator changing your password, be

sure you write down your new password and save it in a secure
place.

To change a user password:

1. Select About > Users.

2. Delete the text in the Password field and type a new password.

3. Click Save.

Restoring an The password file is encrypted and contains all user name and
administrator password information created in the User Logging feature. If you
password forget your administrator password, you can delete the password
file and recreate your administrator account.

Caution! This procedure deletes all user names and

passwords; you must manually recreate each account.

To restore the administrator password:

1. If the C6 Plus software is running, exit the software.

2. Navigate to C:\Cytometer Support Files.

3. Locate the Password file and delete the file.

4. See Signing in to the software (page 252) and Adding user

accounts (page 253) for information about logging in and
creating a new user account.

The administrator user name and password will revert back to

their default settings.

More information • Tracking user activity (page 250)

• Monitoring user activity (page 256)
256 BD Accuri C6 Plus System User’s Guide

Monitoring user activity

Introduction This topic describes how the administrator can monitor user
activity using the userUsage log.

About the Each time a user signs into or out of the software, an entry is made
userUsage log in the userUsage Log. The userUsage log is a CSV file that you can
open using Microsoft Excel or similar spreadsheet program.

The userUsage log contains the following information:

• Date and time of a sign in/sign out

• Username of the operator
• Serial number of the cytometer
• Type of activity (sign in or sign out)
• Notes entered in the User dialog when the user was added
 Chapter 13: User Tracking Module 257

Viewing the To view the userUsage log:

userUsage log 1. Navigate to the C:\Cytometer Support Files folder and open
the folder.

2. Locate userUsage.csv and double-click it to open it.

More information • Tracking user activity (page 250)

• Managing user accounts (page 253)
258 BD Accuri C6 Plus System User’s Guide
 14
Remote Interfacing Module
This chapter covers the following topics:

• Remote Interfacing (page 260)

• Implemented commands (page 262)
• Example usage of remote interfacing commands (page 265)
260 BD Accuri C6 Plus System User’s Guide

Remote Interfacing
Introduction This topic describes the Remote Interfacing Module option and
how to install and use it.

About the Remote The Remote Interfacing Module is an optional module that enables
Interfacing Module the BD Accuri C6 Plus software and BD CSampler Plus software to
receive commands from other software applications via a TCP/IP
connection. Remote Interfacing is designed for advanced users
only.

Once in remote interfacing mode, BD Accuri C6 Plus software

listens through the TCP/IP connection to the XML formatted
commands. Most of these commands execute the same
functionality that you would manually initiate by choosing a menu
item or a button. See Example usage of remote interfacing
commands (page 265) for an example.

Note: The remote interfacing capability is an optional add-on

feature that requires an installed activation key.

Installing the To install the Remote Interfacing Module:

Remote Interfacing 1. Copy the remote interfacing activation key (Remote
Module Interfacing Installer.exe) from the USB drive to the computer
desktop.

2. Double-click the Remote Interfacing Installer icon on the

desktop.

3. In the installation wizard, select the correct directory to install

the activation key.

The location depends on where the C6 Plus software was

installed. In most cases, the location is in one of two places:

• C:\Program Files\BD Accuri\BD Accuri C6 Plus

\ActivationKeys
• C:\Program Files\BD Accuri\BD CSampler
Plus\ActivationKeys
 Chapter 14: Remote Interfacing Module 261

If necessary, use the browse button to navigate to the correct

folder.

4. Click Install.

The software displays a confirmation message after successful

installation.

Setting up remote To set up remote interfacing:

interfacing 1. From BD Accuri C6 Plus software or BD CSampler Plus
software, select Instrument > Remote Interfacing Module. The
C6 software opens a Remote Control dialog box.

The Remote Control dialog box displays a connection string,

which might be needed by the application controlling the C6
Plus or CSampler Plus software. This connection string is the
computer’s IP address with the port number on which the
software will set up a connection and listen for commands.

2. Follow the connection procedure of the third-party software

application that will control the C6 Plus or CSampler Plus
software.

Note: C6 Plus or CSampler software does not queue

commands. The software ignores commands sent to it while it
is busy completing the previous command. Some commands
begin a process that continues even after the software finishes
the command, for example, <CollectSample/>.

Confirming remote To confirm that C6 Plus software or CSampler Plus software is

connection connected to the third-party application:
1. Select Instrument > Remote Interfacing Module. The Remote
Interfacing dialog box appears displaying a connection string
to C6 Plus software or CSampler Plus software (for example,
192.168.0.170:9876). The IP address precedes a colon and the
port number.

2. Use a Telnet client to connect to C6 Plus software or CSampler

Plus software with the connection string. When the connection
is successful, C6 Plus or CSampler Plus software responds with
262 BD Accuri C6 Plus System User’s Guide

<Connected/>.

3. Enter a command such as <ChangeWell wellCode=“B1”/>.

Implemented commands
Introduction This section describes the available remote interfacing commands
to C6 Plus or CSampler Plus software and the return values from
the software.

Commands <AutomaticWellAdvance/>

Description After this command has been sent, whenever the

cytometer is collecting and the 1 million event limit is
reached, collection will spill over into the next well
without loss of data. Close the current C6 Plus
software session and reopen the software to exit this
mode.

Return <OK/>

<ChangeWell wellCode=“A1”/>

Description Changes the currently selected well to the well

indicated in the command.

Return <OK/>

Possible Error <Error>Well does not exist.</Error>

Messages
<Error>Cannot change well while collecting.</Error>

<CollectSample/>

Description Collects data in the currently selected well.

Return <OK/>

Possible Error <Error>The software is currently busy.</Error>

Messages (Software is busy during backflushing and cleaning.)
 Chapter 14: Remote Interfacing Module 263

<NewWorkspace/>

Description Opens a new software workspace.

Return <OK/>

Possible Error <Error>The software is currently busy.</Error>

Messages (Occurs during acquisition.)

<ExportAllAsFCS path=“C:\NewDirectory”/>

Description Exports all FCS files to the specified location.

(This command only requires a file location. The file
name is generated by BD Accuri C6 Plus software.)

Return <OK/>

Possible Error <Error>The software is currently busy.</Error>

Messages (Occurs during acquisition.)
<Error>Files did not save.</Error>
(Occurs if specified root location does not exist;
however, if folder does not exist, it will be created.)

<OpenWorkspace path=“C:\NewDirectory\Filename”/>

Description Opens a new software workspace (requires both a file

location and a file name).

Return <OK/>

Possible Error <Error>The software is currently busy.</Error>

Messages (Occurs during acquisition.)
<Error>File did not open.</Error>
(Occurs if specified file name/location does not exist)
<Error>File name location does not exist.</Error>
<Error>File format not supported.</Error>
<Error>Successfully loaded the event data from your
file, however there were problems loading your
analysis.</Error>
264 BD Accuri C6 Plus System User’s Guide

<Pause/>

Description Pauses acquisition in the currently selected well.

Return <OK/>

Possible Error <Error>The software is currently busy.</Error>

Messages (Software is busy during backflushing and cleaning.)

<ReportStatus/>

Description Whenever this command is sent, C6 Plus or CSampler

Plus software returns a snapshot of the status at the
time the status was requested. This status includes the
current time, hardware status, total event count, and
the status message.

Return <Status><Time>14:28:22.640</Time><Hardware
Status>Running</HardwareStatus><TotalEvent
Count>445785</TotalEventCount><UserMessage>
Events are being recorded.</UserMessage></Status>

<SaveFile path=“C:\NewDirectory\Filename”/>

Description Saves the file to the specified location and name. This
command requires both a file location and file name.
The extension must be included in the file name. To
save the file as a template, the extension is .cit, to
save as a C6 Plus software or CSampler Plus software
workspace file, the extension is .ci.

Return <OK/>

Possible Error <Error>The software is currently busy.<Error>

Messages (Occurs during acquisition.)
<Error>File did not save.</Error>
(Occurs if you do not enter a file location and name.)
 Chapter 14: Remote Interfacing Module 265

Example usage of remote interfacing commands

Introduction This topic provides an example of remote interfacing commands
and responses.

Example For each command sent, a response is sent back.

Command Response
<Connected/>

<OpenWorkspace <OK/>
path=“C:\ExampleFolder\Examplefile.cit”/>

<AutomaticWellAdvance/> <OK/>

<CollectSample/> <OK/>

<ReportStatus/> <Status> <Time>09:28:06.488</Time>

<HardwareStatus>Running</Hardware
Status><TotalEventCount>180223</Total
EventCount><UserMessage>Events are
being recorded.</UserMessage></Status>

Continue using the<ReportStatus/> <OK/>

command until you reach 2,400,000 events.
<Pause/>

<SaveFile <OK/>
path=“C:\NewDirectory\NewFilename.ci”/>
266 BD Accuri C6 Plus System User’s Guide
 15
FCS keywords
This chapter covers the following topics:

• FCS keywords (page 268)

268 BD Accuri C6 Plus System User’s Guide

FCS keywords
Introduction The following tables list the standard and custom keywords used
in FCS files generated by BD Accuri C6 Plus software and
BD CSampler Plus software.

Standard keywords The following table describes the FCS keywords found in FCS files
that are exported by BD Accuri C6 Plus software and
BD CSampler Plus software.

Keyword Description
$FIL Filename including FCS extension.

$WELLID Three-character string representing sample

identifier.

$SMNO Sample name.

$DATATYPE The data type of the actual values for each

event. It is always “I” for unsigned binary
integers.

$MODE The mode of the data. It is always “L” for list

mode where the data is in the order described
by the $Pn keywords.

$BYTEORD Order in which data bytes are written, least to

most significant. It is always 4,3,2,1.

$NEXTDATA The byte offset for an additional data set in

the file. BD C6 Plus files always specify 0 since
the files only contain 1 data set.

$PAR Total number of parameters stored in the data

set. All data sets have 14 parameters.

$PnB For parameter n, the number of bits for each

binary value. The number is always 32 since
integers are stored in 32 bits in Java®.

$PnR The range of parameter n. The range for all

parameters is always 16777216.
 Chapter 15: FCS keywords 269

Keyword Description
$PnN The name of parameter n. Parameters are the
default values from the software.

$PnE For parameter n, this denotes if linear or

logarithmic amplifiers are used. It is always
0,0 because linear values for data are always
saved. This is an optional tag.

$PnS The name of the fluorescence stain used for

parameter n. This tag is used for the custom
parameter name.

$TOT Number of objects stored in the data list. The

cumulative event total for the sample.

$DATE The date the represented sample was last

acquired into DD-MMMYYYY.

$CYT The name of the cytometer used for the

measurement. It is always BD Accuri C6 Plus
flow cytometer.

$CYTSN The serial number of the cytometer used for

the measurement.

$SPILLOVER The standard tag for color compensation.

Refer to the FCS 3.1 specs for a description of
the format.

$TIMESTEP Hard-coded value of 0.1, which is in seconds.

$PROJ Represents the name of the workspace, which

is also the name of the .ci file (before the .ci
extension). If exporting an FCS file, this value
is the name of the file before the FCS
extension.

$BTIM The beginning time of acquisition of the first

event.

$ETIM The end time of acquisition of the last event.

$VOL Total volume in nanoliters.

270 BD Accuri C6 Plus System User’s Guide

Keyword Description
$BEGINSTEXT Default FCS 3.1 tag to mark the beginning of
the text section.

$ENDSTEXT Default FCS 3.1 tag to mark the end of the

text section.

$BEGINANALYSIS Default FCS 3.1 tag to mark the beginning of

the analysis section. Always 0.

$ENDANALYSIS Default FCS 3.1 tag to mark the end of the

analysis section. Always 0.

$BEGINDATA Default FCS 3.1 tag to mark the beginning of

the data section.

$ENDDATA Default FCS 3.1 tag to mark the end of the

data section.

$TR The primary threshold name and value,

separated by a comma.

$PnL Describes the wavelength at which to operate

the parameter’s detector.

Custom keywords The following table describes custom keywords found in FCS files
that are exported by C6 Plus software and CSampler Plus
software. Custom keywords are not required as part of the FCS 3.1
standard.

Keyword Description
#BDACCURIDECADESn The number of decades for
parameter n. It is always
7.224719870049579.

#BDACCURICAPTUREDDATE The date of the most recent

acquisition of the represented
sample, expressed in milliseconds
as a thirteen-character string with
leading zeros.
 Chapter 15: FCS keywords 271

Keyword Description
#ACQUISITIONTIMEMILLI The cumulative run data’s
acquisition time in milliseconds.

#PnVirtualGain The VirtualGain set for parameter

n, where 1.0 means no
VirtualGain.

#SECONDARYTHRESHOLD The secondary threshold name and

value, separated by a comma.

#SAMPLE Value is either the well code or the

sample rename (if one exists).

#ATIM Cumulative acquisition time.

#SPACERS Used to pad the text, analysis, and

data sections of the FCS file. It is
not read and contains no
meaningful data.

#SoftwareVersion A string consisting of

SoftwareFCSName, a space, and
software version.

#PnMaxUsefulDataChannel Only applies when FCS file is

exported. Data value for parameter
n that is the 95th percentile.

#PnDetectorName Name of parameter’s detector.

#PnDimension AREA or HEIGHT, depending on

which attribute was measured.

#PnIsFluorescent True/False value indicating whether

or not parameter measures
fluorescent light.

#PnDetectorNumber Describes the identifying number of

parameter’s detector.

#LASERCONFIGURATION Represents the sample’s laser

settings.
272 BD Accuri C6 Plus System User’s Guide
 16
Technical specifications
This chapter covers the following topics:

• System specifications (page 274)

274 BD Accuri C6 Plus System User’s Guide

System specifications
Introduction This topic describes the system specification.

Cytometer
specifications Item Specification
Dimensions Height: 27.9 cm (11 in.)
Width: 37.5 cm (14.8 in.)
Depth: 41.9 cm (16.5 in.)
With fluid bottles:
Height: 27.9 cm (11 in.)
Width: 54.6 cm (21.5 in.)
Depth: 41.9 cm (16.5 in.)

Weight 13.6 kg (30 lb)

Power Power input requirement:

100–240 VAC, 50/60 Hz
Power supply output:
12 VDC, 11.5 A
Cytometer power:
12 VDC, 120 W (Max)
Heat output 240 BTU/hr maximum output

Operating temperature/ 15–30°C; 15–80% relative humidity

humidity
Laser excitation 488 nm
640 nm

Laser profile Blue laser beam: 9 x 94 µm

Red laser beam: 11 x 104 µm

Laser power 488 nm solid-state blue laser: 20 mW

640 nm diode red laser: 12.5 mW
 Chapter 16: Technical specifications 275

Item Specification
Emission detection 4 colors, standard optical filters
 FL1 533/30 nm
 FL2 585/40 nm
 FL3 >670 nm
 FL4 675/25 nm

Optical alignment Fixed alignment

Flow cell 200-µm ID quartz capillary

Minimum detectable 0.5 µm

particle size
Minimum sample volume 50 µL (12 x 75-mm tubes)
150 µL (BD Trucount tubes)
100 µL (CSampler Plus, 12 x 75-mm
tubes)

Flow rate 14–66 µL/min, depending on the test

Maximum events/sample 1 million events

Fluorescence sensitivity FITC <75

MESF*
PE <50

Fluorescence linearity 2 ±0.05% for chicken erythrocyte nuclei

(CEN)

Fluorescence precision ≤3% CV for CEN

Data acquisition rate 10,000 events/s, maximum

Fluid bottle capacity 2 L sheath, 2 L waste

250 mL BD FACSClean
250 mL Detergent Solution
276 BD Accuri C6 Plus System User’s Guide

Item Specification
Signal processing 24-bit datapath

Computer interface USB 2.0 or higher

* MESF values determined using Thermo Scientific Cyto-Cal™

Multifluor Plus Violet Beads.

Computer
specifications Item Specification
General PC desktop workstation with removable
SD card

Dimensions (without Height: 38.1 cm (15 in.)

stand)
Width: 56.8 cm (22.3 in.)
Depth: 6.0 cm (2.4 in.)

Weight 9.1 kg (20.1 lb)

Processor 64-bit multi-core processor

Operating system Microsoft Windows® 7

Software Windows 7 Professional and BD Accuri

C6 Plus software or BD CSampler Plus
software

Storage 26 GB free hard disk space after operating

system and software application
installation

RAM 8 GB

USB ports USB 3.0 - 2 side/4 rear

USB 2.0 - 2 rear

Accessories Windows-ready mouse and keyboard

 Chapter 16: Technical specifications 277

Optional CSampler
Plus Item Specification
Dimensions Length: 50.8 cm (20 in.)
Width: 35.6 cm (14 in.)
Height: 20.3 cm (8 in.)

Weight 3.63 kg (8 lb)

Connection Serial

Tray Includes three fixed tube locations for

cleaning solutions

Compatible racks Custom 24-tube capacity

Compatible plates  96-well U-bottom

 96-well flat bottom
 96-well V-bottom
 96 deep well
 48 well
278 BD Accuri C6 Plus System User’s Guide

Optional barcode The optional 2D barcode reader, a Motorola® DS6707-HC

reader handheld imager, connects to computer through a USB connection,
and reads the following barcode standards.

Item Specification
Standards  Code 39
 Codabar
 Code 128
 Interleaved 2 or 5
 Universal Product Code (UPC)
 Maxicode
 DataMatrix
 PDF417
 Index
A barcode reader 14, 278
acquisition batch analysis 142–144
auto collect 112–115 BD Accuri C6 Plus system
collecting precise volumes 214–217 See also cytometer
CSampler Plus 93–96 overview 14, 16–22
manual 81–83 BD FACSClean
parameters, setting 72 bottle 21
troubleshooting 229 filling 38
workflow, auto collect 102 beads
workflow, CSampler Plus 92 See CS&T RUO beads
workflow, manual 72 bottle
adjusting BD FACSClean 21
height of sample stage 23 detergent solution 21
peak position with VirtualGain 134 filling 38
QC markers 54–56 filters, replacing 203–204
sample stream core 76 sheath 21
volume settings 215 waste 21
agitating samples 96, 108
analysis C
batch 142–144 C6 Plus system
sample data file 123 See also cytometer
setting up plots 124–125 overview 14, 16–22
statistics 130 Clean Fluidics cycle 200
using VirtualGain 135–140 cleaning
viewing data 125 backflushing SIP 197
auto-adjust volume settings 215 Clean Fluidics cycle 200
auto-saving data 186 extended flow cell clean 200–201
outside of instrument 196
B sample stage 43
backflushing SIP 197 SIP clean 198–199
tubes, in CSampler Plus rack 26
280 BD Accuri C6 Plus System User’s Guide

color gating 172–174 cytometer power

compensation button 17
adjusting 149–153 connector 18
controls 147 indicator 17
preferences 148
computer D
overview 15
daily
specifications 276
maintenance 194
connector
QC workflow 48
CSampler Plus 18
shutdown 43–45
power 18
startup 41–42
CS&T RUO beads
startup workflow 36
deleting old lots 185
data
installing new lot 51
acquisition, auto collect 112–115
running 49–51
acquisition, CSampler Plus 93–96
CSampler Plus
acquisition, manual 81–83
aligning after collision 217–218
adding more to files 83, 96
cleaning tube locations 26
manually saving 187
connector 18
detergent solution
loading rack 25
bottle 21
overview 24–27
filling 38
specifications 277
downloading CS&T RUO bead lot 51
tips for use 27
cytometer
See also maintenance E
event indicator 17 emptying waste 39
fluid bottles 21 enlarging plots 56
fluidics components 20–22 entering CS&T RUO bead lot 51
lasers 20 event indicator 17
optical components 19 exporting
optical filters 20 data 190
overview 14, 16–22 from batch analysis tab 145
pumps 22 extended flow cell clean 200–201
restarting after storage 219
sample stage 17 F
shutdown 43–45
FACSClean
SIP 17, 22
bottle 21
specifications 274–276
filling 38
startup 41–42
files
startup workflow 36
exporting 190
 Index 281

importing 191 H
managing 184–185 histogram overlay 127–129
sample analysis file 123
saving 187
workspace 186–188 I
filter importing data 191
fluid bottle, replacing 203–204 indicator
in-line sheath 22, 204–206 event 17
optical 20 power 17
fluid bottles in-line sheath filter
emptying waste 39 overview 22
filling 38 replacing 204–206
fluid sensing line 204 installing
overview 21 CS&T RUO bead lot 51
replacing filters 203–204 sample tube 22
fluidics selectable lasers module 237
cleaning 200 user tracking module 250
components, overview 20–22
harness 18 K
tubing, replacing 206–209 keywords
fluidics rate custom 270
customizing 76 standard 268
setting 75
fluids required 37
fluorescence spillover L
See also compensation lasers 20
about 146–147 Levey-Jennings report
correcting 149–153 printing 65
viewing 63
live gates 166
G
loading
gates 24-tube rack 25
See also regions sample tube 22
applying to plot 164
color gating 172–174
creating 161 M
deleting 164 maintenance
live 166 See also cleaning
nested 168–170 aligning CSampler Plus 217–218
types 160–164 Clean Fluidics cycle 200
daily 194
282 BD Accuri C6 Plus System User’s Guide

extended flow cell clean 200–201 QC 59

replacing fluid bottle filters 203–204 renaming 160
replacing in-line sheath filter 204– renaming axes 177
206 setting up for analysis 124–125
replacing pump tubing 206–209 specifications, changing 158
scheduled 194–195 viewing data in 125
SIP clean 198–199 zooming 56, 179
unclogging SIP 211–214 zooming in 177
unscheduled 195 port, USB 18
wiping instrument 196 power
managing user accounts button 17
See user tracking module connector 18
indicator 17
O printing
Levey-Jennings report 65
optical
plots 182
components 19
QC report 62
filters 20
statistics 182
selectable lasers filters 236
pump
overview
sheath 22
CSampler Plus 24–27
tubing, replacing 206–209
cytometer 16–22
waste 22
fluid bottles 21
QC 48
software 28–34 Q
QC
P failed result 57
messages 60
parameters, changing plot 176
module 48
PC
overview 48
overview 15
results 58–61
specifications 276
running 49–51
plot tools 156
troubleshooting 231
plots
workflow 48
changing number of events 175
QC report
changing parameter 176
adding comments 61
copying and printing 181
adjusting markers 54–56
creating new 127, 157
deleting from history list 184
format for drag/drop 181
plots 59
histogram overlay 127–129
printing 62
printing 182
results table 59
 Index 283

viewing previous 61 sample tube

zooming plots 56 loading 22
specifications 15
R saving
workspace files 187
rack
selectable lasers
loading 25
about 236
map 26
annotating files 238
specifications 24
application examples 243–246
reagents 15
components 236
regions
configurations 240
adjusting QC 54–56
installing 237
coloring 172–174
proper filter placement 240
moving/resizing 171
setting
renaming 174
acquisition parameters 72
replacing
fluidics rate 75
fluid bottle filters 203–204
run direction 109
in-line sheath filter 204–206
run limits 73–74
peristaltic pump tubing 206–209
threshold 80
report
sheath
Levey-Jennings 63
bottle 21
QC 58–61
filling 38
results
filter, in-line 22, 204–206
QC 58–61
pump 22
run direction, setting 109
shutting down 43–45
run limits, setting 73–74
SIP 17, 22
backflushing 197
S clean cycle 198–199
sample analysis file 123 unclogging 211–214
sample injection probe software
See SIP overview 28–34
sample sets troubleshooting 227
creating for auto collect 103–105 starting up 36, 41–42
sample stage statistics
adjusting height 23 master table 131
cleaning 43 previewing plots in tab 132
loading tubes 22 tab 130
overview 17 stopping criteria, setting 73–74
sample stream
adjusting core size 76
284 BD Accuri C6 Plus System User’s Guide

T emptying 39
template, workspace 189 pump 22
threshold workflow
about 79 auto collect 102
setting 80 CSampler Plus acquisition 92
troubleshooting manual acquisition 72
acquisition 229 QC 48
hardware 222 startup 36
QC 231 workspace
software 227 C6 Plus 29–30
tube rack creating templates 189
loading 25 CSampler Plus 30–31
map 26 files 186–188
specifications 24 opening new 188
tubes workstation
loading 22 overview 15
specifications 15 specifications 276
tubing, replacing 206–209
Z
U zooming plots 56, 179
USB port 18
user tracking module
installing 250
managing user accounts 253
monitoring user activity 256
signing in 252

V
VirtualGain
applying 135–140
changing views 141
purpose 134
removing 142
viewing 140

W
waste
bottle 21