CD90 MicroBeads

human

Order no. 130-096-253

Contents 1.1 Principle of the MACS® Separation

1. Description First, the CD90+ cells are magnetically labeled with CD90
1.1 Principle of the MACS® Separation MicroBeads. Then, the cell suspension is loaded onto a MACS®
Column, which is placed in the magnetic field of a MACS Separator.
1.2 Background information The magnetically labeled CD90+ cells are retained within the
1.3 Applications column. The unlabeled cells run through; this cell fraction is thus
depleted of CD90+ cells. After removing the column from the
1.4 Reagent and instrument requirements
magnetic field, the magnetically retained CD90+ cells can be eluted
2. Protocol as the positively selected cell fraction. To increase the purity, the
2.1 Sample preparation positively selected cell fraction containing the CD90+ cells must be
separated over a second column.
2.2 Magnetic labeling
2.3 Magnetic separation 1.2 Background information
2.4 
Cell separation with the autoMACS® Pro Separator The CD90 antibody reacts with the human CD90 antigen, also
3. Example of a separation using the CD90 MicroBeads known as Thy-1, a 25–35 kD GPI-anchored protein of the Ig
superfamily involved in cell-cell and cell-matrix interactions.
4. References CD90 was identified as a marker for cancer stem cells in human
liver cancer.¹ In addition to cancer stem cells, it is expressed on
embryonic stem cells (ESC), induced pluripotent stem cells (iPSC),
Warnings neurons, small subsets of human fetal liver cells and thymocytes,
Reagents contain sodium azide. Under acidic conditions sodium cord blood, and bone marrow cells. CD90 is also expressed on a
azide yields hydrazoic acid, which is extremely toxic. Azide subset of CD34+ primitive hematopoietic stem cells. CD90+CD34+
compounds should be diluted with running water before discarding. cells characterize a highly enriched population of high proliferative
These precautions are recommended to avoid deposits in plumbing potential colony-forming cells (HPP-CFC).² Furthermore, CD90
where explosive conditions may develop. expression is found on mesenchymal stem cells (MSCs).³

1.3 Applications
1. Description
● Positive selection or depletion of cells expressing human
This product is for research use only.
CD90 antigen, e.g., liver cancer stem cells.
Components 2 mL CD90 MicroBeads, human:
MicroBeads conjugated to monoclonal anti- 1.4 Reagent and instrument requirements
human CD90 antibodies (mouse IgG1).
● Buffer: Prepare a solution containing phosphate-buffered
Capacity For 10⁹ total cells, up to 100 separations.
saline (PBS), pH  7.2, 0.5% bovine serum albumin
Product format CD90 MicroBeads are supplied in buffer (BSA), and 2 mM EDTA by diluting MACS® BSA Stock
containing stabilizer and 0.05% sodium azide. Solution (#  130‑091‑376) 1:20 with autoMACS® Rinsing
Storage Store protected from light at 2−8 °C. Do not Solution (# 130‑091‑222). Keep buffer cold (2−8 °C).
freeze. The expiration date is indicated on the Always use freshly prepared buffer. Do not use autoMACS
vial label. Running Buffer or MACSQuant® Running Buffer as they
contain a small amount of sodium azide that could affect the
results.
▲▲ Note: EDTA can be replaced by other supplements such as anticoagulant
citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA
can be replaced by other proteins such as human serum albumin, human serum,
or fetal bovine serum (FBS). Buffers or media containing Ca 2+ or Mg 2+ are not
recommended for use.

Miltenyi Biotec GmbH Miltenyi Biotec Inc.

140-003-333.03

Friedrich-Ebert-Straße 68, 51429 Bergisch Gladbach, Germany 2303 Lindbergh Street, Auburn, CA 95602, USA
Phone +49 2204 8306-0, Fax +49 2204 85197 Phone 800 FOR MACS, +1 530 888 8871, Fax +1 877 591 1060
[email protected] [email protected]
www.miltenyibiotec.com
page 1/4
 Order no. 130-096-253

● MACS Columns and MACS Separators: CD90+ cells can be

enriched by using MS or LS Columns or depleted with the
use of LD Columns. Cells that strongly express the CD90 2.2 Magnetic labeling
antigen can also be depleted using MS or LS Columns. Positive
selection or depletion can also be performed by using the ▲ Cells can be labeled with MACS MicroBeads using the
autoMACS Pro Separator. autolabeling function of the autoMACS Pro Separator. For more
Column  Max. Max. number Separator
information refer to section 2.4.
number of of total cells
labeled cells ▲ Work fast, keep cells cold, and use pre-cooled solutions. This will
prevent capping of antibodies on the cell surface and non-specific
Positive selection
cell labeling.
MS 10⁷ 2×10⁷ MiniMACS, OctoMACS,
VarioMACS, SuperMACS II ▲ Volumes for magnetic labeling given below are for up to
10⁷ total cells. When working with fewer than 10⁷ cells, use the same
LS 2×10⁷ 4×10⁷ MidiMACS, QuadroMACS,
VarioMACS, SuperMACS II volumes as indicated. When working with higher cell numbers,
scale up all reagent volumes and total volumes accordingly (e.g.
Depletion
for 2×10⁷ total cells, use twice the volume of all indicated reagent
LD 1.5×10⁷ 3×10⁷ MidiMACS, QuadroMACS, volumes and total volumes).
VarioMACS, SuperMACS II
▲ For optimal performance it is important to obtain a single‑cell
Positive selection or depletion suspension before magnetic labeling. Pass cells through 30 µm
autoMACS 5×10⁷ 10⁸ autoMACS Pro nylon mesh (Pre-Separation Filters (30 µm) # 130-041-407) to
remove cell clumps which may clog the column. Moisten filter with
▲▲  Note: Column adapters are required to insert certain columns into the buffer before use.
VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective
MACS Separator data sheet.
▲ The recommended incubation temperature is 2–8 °C. Higher
temperatures and/or longer incubation times may lead to
● (Optional) Fluorochrome-conjugated CD90 antibodies for non‑specific cell labeling. Working on ice may require increased
flow cytometric analysis, e.g., CD90-PE and CD326 (EpCAM)- incubation times.
APC. For more information about antibodies refer to
www.miltenyibiotec.com/antibodies. 1. Determine cell number.
● (Optional) Propidium Iodide Solution (# 130-093-233) or 2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate
7-AAD for flow cytometric exclusion of dead cells. supernatant completely.
● (Optional) Dead Cell Removal Kit (# 130-090-101) for the 3. Resuspend cell pellet in 80 µL of buffer per 10⁷ total cells.
depletion of dead cells.
4. Add 20 µL of CD90 MicroBeads per 10⁷ total cells.
● (Optional) Pre-Separation Filters (30 µm) (# 130-041-407) to
remove cell clumps. 5. Mix well and incubate for 15 minutes in the dark in the
refrigerator (2−8 °C).
2. Protocol 6. (Optional) Add staining antibodies, e.g., 10 µL of CD90-PE,
and incubate for 5 minutes in the dark in the refrigerator
2.1 Sample preparation
(2−8 °C).
When working with anticoagulated peripheral blood or buffy coat,
7. Wash cells by adding 1−2 mL of buffer per 10⁷ cells and
peripheral blood mononuclear cells (PBMCs) should be isolated by
centrifuge at 300×g for 10 minutes. Aspirate supernatant
density gradient centrifugation, for example, using Ficoll-Paque™.
completely.
▲▲  Note: To remove platelets after density gradient separation, resuspend cell
pellet in buffer and centrifuge at 200×g for 10−15 minutes at 20 °C. Carefully 8. Resuspend up to 10⁸ cells in 500 µL of buffer.
aspirate supernatant. Repeat washing step. ▲ Note: For higher cell numbers, scale up buffer volume accordingly.
▲ Note: For depletion with LD Columns, resuspend up to 1.25×10⁸ cells in
When working with lysed blood, prepare a single-cell suspension 500 µL of buffer.
using standard methods.
For details refer to the protocols section at www.miltenyibiotec.com/ 9. Proceed to magnetic separation (2.3).
protocols.
When working with tissues, prepare a single-cell suspension using
the gentleMACS™ Dissociator.
For details refer to www.gentlemacs.com/protocols.
▲ Dead cells may bind non-specifically to MACS MicroBeads.
To remove dead cells, we recommend using density gradient
centrifugation or the Dead Cell Removal Kit (# 130-090-101).

Unless otherwise specifically indicated, Miltenyi Biotec products and services page 2/4
140-003-333.03

are for research use only and not for diagnostic or therapeutic use.
 Order no. 130-096-253

2.4 Cell separation with the autoMACS® Pro Separator

2.3 Magnetic separation ▲ Refer to the user manual for instructions on how to use the
autoMACS® Pro Separator.
▲ Choose an appropriate MACS Column and MACS Separator ▲ All buffer temperatures should be ≥10 °C.
according to the number of total cells and the number of CD90+ cells.
▲ For appropriate resuspension volumes and cell concentrations,
For details refer to the table in section 1.4.
please visit www.automacspro.com/autolabeling.
▲ Always wait until the column reservoir is empty before
▲ Place tubes in the following Chill Rack positions:
proceeding to the next step.
position A = sample, position B = negative fraction,
Magnetic separation with MS or LS Columns position C = positive fraction.
1. Place column in the magnetic field of a suitable MACS
Separator. For details refer to the respective MACS Column 2.4.1 F
 ully automated cell labeling and separation
data sheet.
1. Switch on the instrument for automatic initialization.
2. Prepare column by rinsing with the appropriate amount of
2. Go to the Reagent menu and select Read Reagent. Scan the
buffer:
2D barcode of each reagent vial with the barcode scanner
MS: 500 µL LS: 3 mL on the autoMACS Pro Separator. Place the reagent into the
3. Apply cell suspension onto the column. Collect flow-through appropriate position on the reagent rack.
containing unlabeled cells. 3. Place sample and collection tubes into the Chill Rack.
4. Wash column with the appropriate amount of buffer. Collect 4. Go to the Separation menu and select the reagent name for
unlabeled cells that pass through and combine with the each sample from the Labeling submenu (the correct labeling,
flow‑through from step 3. separation, and wash protocols will be selected automatically).
MS: 3×500 µL LS: 3×3 mL 5. Enter sample volume into the Volume submenu. Press Enter.
▲ Note: Perform washing steps by adding buffer aliquots as soon as the column 6. Select Run.
reservoir is empty.

5. Remove column from the separator and place it on a suitable 2.4.2 Magnetic separation with the autoMACS® Pro Separator
collection tube. using manual labeling

6. Pipette the appropriate amount of buffer onto the column. 1. Label the sample as described in section 2.2 Magnetic labeling.
Immediately flush out the magnetically labeled cells 2. Prepare and prime the instrument.
by firmly pushing the plunger into the column.
3. Apply tube containing the sample and provide tubes for
MS: 1 mL LS: 5 mL collecting the labeled and unlabeled cell fractions. Place
7. (Optional) To increase the purity of CD90+ cells, the eluted sample tube in row A of the tube rack and the fraction
fraction can be enriched over a second MS or LS Column. collection tubes in rows B and C.
Repeat the magnetic separation procedure as described in 4. For a standard separation choose the following program:
steps 1 to 6 by using a new column.
Positive selection: Posseld2
Collect positive fraction in row C of the tube rack.
Depletion with LD Columns
Depletion: Deplete
1. Place LD Column in the magnetic field of a suitable MACS Collect negative fraction in row B of the tube rack.
Separator. For details refer to the LD Column data sheet.
2. Prepare column by rinsing with 2 mL of buffer.
3. Apply cell suspension onto the column.
4. Collect unlabeled cells that pass through and wash
column with 2×1 mL of buffer. Collect total flow-through;
this is the unlabeled cell fraction. Perform washing
steps by adding buffer two times. Only add new buffer when
the column reservoir is empty.

Unless otherwise specifically indicated, Miltenyi Biotec products and services page 3/4
140-003-333.03

are for research use only and not for diagnostic or therapeutic use.
 Order no. 130-096-253

3. Example of a separation using CD90 MicroBeads Warranty

The products sold hereunder are warranted only to be free from defects in workmanship
CD90+ human teratocarcinoma cells (NTERA2) were isolated from and material at the time of delivery to the customer. Miltenyi Biotec GmbH
a mixture of U937 and NTERA2 cells using CD90 MicroBeads, makes no warranty or representation, either expressed or implied, with respect to
the fitness of a product for a particular purpose. There are no warranties, expressed
an MS Column, and an OctoMACS™ Separator. Cells were
or implied, which extend beyond the technical specifications of the products.
fluorescently stained with CD90-PE and CD326 (EpCAM)-APC Miltenyi Biotec GmbH’s liability is limited to either replacement of the products or
and analyzed by flow cytometry using the MACSQuant® Analyzer. refund of the purchase price. Miltenyi Biotec GmbH is not liable for any property
Cell debris and dead cells were excluded from the analysis based on damage, personal injury or economic loss caused by the product.
scatter signals and propidium iodide fluorescence.
autoMACS, gentleMACS, MACS, MACSQuant, MidiMACS, MiniMACS,
OctoMACS, QuadroMACS, SuperMACS, and VarioMACS are registered trademarks
Before separation
or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries
10³ worldwide.
CD326 (EpCAM)-APC

10² Ficoll-Paque is a trademark of GE Healthcare companies.

10¹ Copyright © 2016 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved.

1
0
-1
-1 0 1 10¹ 10² 10³
CD90-PE

CD90+ cells

4. References
1. Zhen Fan Yang, Z. F. et al. (2008) Significance of CD90+ Cancer Stem Cells in
Human Liver Cancer. Cancer Cell 13: 153–166.
2. Mayani, H. and Lansdorp, P. M. et al. (1994) Thy-1 expression is linked to
functional properties of primitive hematopoietic progenitor cells from human
umbilical cord blood. Blood 83: 2410–2417.
3. Dominici, M. et al. (2006) Minimal criteria for defining multipotent
mesenchymal stromal cells. The International Society for Cellular Therapy
position statement. Cytotherapy 8: 315–317.

Refer to www.miltenyibiotec.com for all data sheets and protocols.

Unless otherwise specifically indicated, Miltenyi Biotec products and services page 4/4
140-003-333.03

are for research use only and not for diagnostic or therapeutic use.