CD34 MicroBead Kit

human
2 mL 130-046-702
10 mL 130-046-703

Contents 1.1 Principle of the MACS Separation

1. Description First, the CD34+ cells are magnetically labeled with CD34
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 CD34+ cells are retained within the
1.3 Applications column. The unlabeled cells run through; this cell fraction is thus
depleted of CD34+ cells. After removing the column from the
1.4 Reagent and instrument requirements
magnetic field, the magnetically retained CD34+ cells can be eluted
2. Protocol as the positively selected cell fraction.
2.1 Sample preparation
1.2 Background information
2.2 Magnetic labeling
The CD34 antigen is a single chain transmembrane glycoprotein
2.3 Magnetic separation
expressed on human hematopoietic progenitor cells, endothelial
2.4 (Optional) Evaluation of hematopoietic progenitor progenitor cells, vascular endothelial cells, embryonic fibroblasts,
cell purity and some cells in fetal and adult nervous tissue.
3. Example of a separation using the CD34 MicroBead Kit The CD34 MicroBead Kit contains MicroBeads directly conjugated
4. References to CD34 antibodies for magnetic labeling of CD34-expressing cells
from peripheral blood, cord blood, bone marrow, apheresis harvest,
or differentiated ES and iPS cells. Hematopoietic progenitor cells,
present at a frequency of about 0.050.2% in peripheral blood,
1. Description 0.10.5% in cord blood, and 0.53% in bone marrow, can be rapidly
Components 2 mL CD34 MicroBeads, human: and efficiently enriched.
MicroBeads conjugated to monoclonal mouse
anti-human CD34 antibodies (isotype: mouse 1.3 Applications
IgG1).
Positive selection or depletion of cells expressing human CD34
2 mL FcR Blocking Reagent, human:
antigen.,
Human IgG.
Isolation of hematopoietic progenitor cells.
or
Isolation of endothelial progenitor cells (EPCs).,
10 mL CD34 MicroBeads, human:
MicroBeads conjugated to monoclonal mouse Isolation of CD34+ progenitor cells from differentiated ES and
anti-human CD34 antibodies (isotype: mouse iPS cell cultures.
IgG1). In vitro differentiation studies.,
10 mL FcR Blocking Reagent, human: Studies on hematologic malignancies.
Human IgG.
Capacity 2 mL: For 210 total cells, up to 20 separations. 1.4 Reagent and instrument requirements
or Buffer: Prepare a solution containing phosphate-buffered saline
10 mL: For 10 total cells, up to 100 separations. (PBS), pH7.2, 0.5% bovine serum albumin (BSA), and 2mM
Product format CD34 MicroBeads are supplied in buffer EDTA by diluting MACS BSA Stock Solution (#130091376)
containing stabilizer and 0.05% sodium azide. 1:20 with autoMACS Rinsing Solution (#130091222). Keep
buffer cold (28C). Degas buffer before use, as air bubbles
Storage Store protected from light at 28C. Do not
could block the column.
freeze. The expiration date is indicated on the
Note: EDTA can be replaced by other supplements such as anticoagulant
vial label.
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.
140-001-950.05

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Friedrich-Ebert-Strae 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 +1 530 888 8925
[email protected] [email protected]
www.miltenyibiotec.com
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 Order no. 130-046-702
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MACS Columns and MACS Separators: CD34+ cells can be Preparation of cells from leukapheresis material
enriched by using MS, LS, or XS Columns (positive selection).
1. Filter apheresis harvest through 30m nylon mesh (Pre-
Cells that strongly express the CD34 antigen can also be
Separation Filters, 30 m #130-041-407), in order to remove
depleted using MS, LS, or XS Columns. Positive selection or
cell clumps.
depletion can also be performed by using the autoMACS Pro
or the autoMACS Separator. 2. Wash cells once with buffer and resuspend in a final volume
of 300L of buffer for up to 10 cells. Proceed to magnetic
Column Max. number Max. number Separator labeling.
of labeled cells of total cells

Positive selection
MS 10 210 MiniMACS, OctoMACS, 2.2 Magnetic labeling
VarioMACS, SuperMACSII
LS 10 210 MidiMACS, QuadroMACS, Work fast, keep cells cold, and use pre-cooled solutions. This will
VarioMACS, SuperMACSII prevent capping of antibodies on the cell surface and non-specific
XS 10 210 SuperMACSII cell labeling.

Positive selection Volumes for magnetic labeling given below are for up to
10 total cells. When working with fewer than 10 cells, use the same
autoMACS 210 410 autoMACS Pro, autoMACS
volumes as indicated. When working with higher cell numbers,
Note: Column adapters are required to insert certain columns into the scale up all reagent volumes and total volumes accordingly (e.g.
VarioMACS or SuperMACS II Separators. For details refer to the respective for 210 total cells, use twice the volume of all indicated reagent
MACS Separator data sheet. volumes and total volumes).
(Optional) MC CD34 Stem Cell Cocktail (#130-093-427) for For optimal performance it is important to obtain a singlecell
flow cytometric analysis of separated cells. suspension before magnetic labeling. Pass cells through 30m
nylon mesh (Pre-Separation Filters, 30 m #130-041-407) to
(Optional) Fluorochrome-conjugated antibodies for flow remove cell clumps which may clog the column. Moisten filter with
cytometric analysis, e.g., CD34-FITC (# 130-081-001), buffer before use.
CD34-PE (# 130-081-002), CD34-APC (# 130-090-954),
The recommended incubation temperature is 28C. Higher
CD133 (293C3)-PE (# 130-090-853), CD133 (293C3)-PE
temperatures and/or longer incubation times may lead to non-
(#130-090-854), CD45-FITC (#130-080-202), CD45-PE
specific cell labeling. Working on ice may require increased
(#130-080-201), or CD45-APC (#130-091-230). For more
incubation times.
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 300g for 10minutes. 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 300 L of buffer for up to 10 total
depletion of dead cells.
cells.
(Optional) Pre-Separation Filters, 30 m (#130-041-407) to
4. Add 100 L of FcR Blocking Reagent for up to 10 total cells.
remove cell clumps.
5. Add 100L of CD34 MicroBeads for up to 10 total cells.
6. Mix well and incubate for 30minutes in the refrigerator
2. Protocol (28C).
2.1 Sample preparation 7. (Optional) Add fluorochrome-conjugated CD34 antibody
When working with anticoagulated peripheral blood or buffy coat, recognizing another epitope than QBEND/10 (e.g. clone
peripheral blood mononuclear cells (PBMCs) should be isolated by AC136: CD34-PE, #130-081-002) or fluorochrome-conjugated
density gradient centrifugation, for example, using Ficoll-Paque. CD45 antibody (e.g. CD45-FITC, # 130-080-202), and
Note: To remove platelets after density gradient separation, resuspend cell
incubate for 5minutes in the dark in the refrigerator (28C).
pellet in buffer and centrifuge at 200g for 1015minutes at 20C. Carefully 8. Wash cells by adding 510mL of buffer for up to 10 cells
aspirate supernatant. Repeat washing step.
and centrifuge at 300g for 10minutes. Aspirate supernatant
When working with tissues or lysed blood, prepare a single-cell completely.
suspension using standard methods. 9. Resuspend up to 10 cells in 500 L of buffer.
For details refer to the protocols section at www.miltenyibiotec.com/ Note: For higher cell numbers, scale up buffer volume accordingly.
protocols. Note: For depletion with LD Columns, resuspend up to 1.2510 cells in
500L of buffer.
Dead cells may bind non-specifically to MACS MicroBeads.
To remove dead cells, we recommend using density gradient 10. Proceed to magnetic separation (2.3).
centrifugation or the Dead Cell Removal Kit (#130-090-101).
140-001-950.05

Unless otherwise specifically indicated, Miltenyi Biotec

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

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 Order no. 130-046-702
Order no. 130-046-703

Magnetic separation with the autoMACS Pro Separator

2.3 Magnetic separation 1. Prepare and prime the instrument.
2. Apply tube containing the sample and provide tubes for
Choose an appropriate MACS Column and MACS Separator collecting the labeled and unlabeled cell fractions. Place
according to the number of total cells and the number of CD34+ cells. sample tube in row A of the tube rack and the fraction
For details refer to the table in section 1.4. collection tubes in rows B and C.
Always wait until the column reservoir is empty before
proceeding to the next step. 3. For a standard separation choose one of the following
programs:
Magnetic separation with MS or LS Columns Positive selection of CD34+ cells from peripheral blood,
1. Place column in the magnetic field of a suitable MACS bone marrow or leukapheresis: Posseld.
Separator. For details refer to the respective MACS Column Positive selection of CD34+ cells from cord blood:
data sheet. Posseld2.
Collect positive fraction in row C of the tube rack.
2. Prepare column by rinsing with the appropriate amount of
buffer:
Magnetic separation with the autoMACS Separator
MS: 500L LS: 3mL
1. Prepare and prime the instrument.
3. Apply cell suspension onto the column. Collect flow-through
containing unlabeled cells. 2. Apply tube containing the sample and provide tubes for
collecting the labeled and unlabeled cell fractions. Place
4. Wash column with the appropriate amount of buffer. Collect sample tube at the uptake port and the fraction collection
unlabeled cells that pass through and combine with the flow- tubes at port neg1 and port pos2.
through from step 3.
3. For a standard separation choose one of the following
MS: 3500L LS: 33mL programs:
Note: Perform washing steps by adding buffer aliquots only when the column Positive selection of CD34+ cells from peripheral blood,
reservoir is empty. bone marrow or leukapheresis: Posseld.
5. Remove column from the separator and place it on a suitable Positive selection of CD34+ cells from cord blood:
collection tube. Posseld2.
Collect positive fraction from outlet port pos2.
6. Pipette the appropriate amount of buffer onto the column.
Immediately flush out the magnetically labeled cells
by firmly pushing the plunger into the column. 2.4 (Optional) Evaluation of hematopoietic progenitor cell
MS: 1mL LS: 5mL purity

7. (Optional) To increase the purity of CD34+ cells, the eluted The purity of the isolated hematopoietic progenitor cells can be
fraction can be enriched over a second MS or LS Column. evaluated by flow cytometry or fluorescence microscopy. Analysis
Repeat the magnetic separation procedure as described in of CD34+ cells can be accomplished by direct immunofluorescent
steps 1 to 6 by using a new column. staining using an antibody recognizing an epitope different from
that recognized by the CD34 monoclonal antibody QBEND/10 (e.g.
Magnetic separation with XS Columns CD34-PE, clone: AC136, #130-081-002).
For optimal discrimination of CD34+ cells from other leukocytes,
For instructions on the column assembly and the separation refer counterstain cells with an antibody against CD45 (e.g. CD45FITC,
to the XS Column data sheet. #130-080-202). CD34+ cells express CD45 at a lower level as
compared to lymphocytes.
Magnetic separation with the autoMACS Pro Separator Use the antibodies in appropriate concentrations as recommended
or the autoMACS Separator by the manufacturers. Typically, staining for 5minutes at 28C
Refer to the respective user manual for instructions on how to should be sufficient. After fluorescent staining, cells should be
use the autoMACS Pro Separator or the autoMACS Separator. washed and resuspended in buffer.

Buffers used for operating the autoMACS Pro Separator or the

autoMACS Separator should have a temperature of 10C.
Program choice depends on the isolation strategy, the strength
of magnetic labeling, and the frequency of magnetically labeled
cells. For details refer to the section describing the cell separation
programs in the respective user manual.
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Unless otherwise specifically indicated, Miltenyi Biotec

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

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 Order no. 130-046-702
Order no. 130-046-703

3. Example of a separation using the Warnings

Reagents contain sodium azide. Under acidic conditions sodium azide yields
CD34 MicroBead Kit hydrazoic acid, which is extremely toxic. Azide compounds should be diluted with
running water before discarding. These precautions are recommended to avoid
Isolation of CD34+ cells from PBMCs using the CD34 MicroBead Kit,
deposits in plumbing where explosive conditions may develop.
two MS Columns, and a MiniMACS Separator. Cells were stained
with CD34-PE (#130-081-002) and CD45-FITC (#130080-202). Warranty
Cell debris and dead cells are excluded from the analysis based on The products sold hereunder are warranted only to be free from defects in workmanship
scatter signals and propidium iodide fluorescence. and material at the time of delivery to the customer. Miltenyi Biotec GmbH
makes no warranty or representation, either expressed or implied, with respect to
Before separation the fitness of a product for a particular purpose. There are no warranties, expressed
or implied, which extend beyond the technical specifications of the products.
Miltenyi Biotec GmbHs liability is limited to either replacement of the products or
refund of the purchase price. Miltenyi Biotec GmbH is not liable for any property
damage, personal injury or economic loss caused by the product.
CD45-FITC

autoMACS and MACS are registered trademarks and MidiMACS, MiniMACS,

OctoMACS, QuadroMACS, SuperMACS, and VarioMACS are trademarks of
Miltenyi Biotec GmbH.

Ficoll-Paque is a trademark of GE Healthcare companies.

CD34-PE
Copyright 2013 Miltenyi Biotec GmbH. All rights reserved.
+
CD34 cells
CD45-FITC

CD34-PE

4. References
1. Giarratana, M. C. et al. (2005) Ex vivo generation of fully mature human red
blood cells from hematopoietic stem cells. Nat. Biotechnol. 23: 6974.
2. Timmermans, F. et al. (2007) Endothelial outgrowth cells are not derived from
CD133+ cells or CD45+ hematopoietic precursors. Arterioscler. Thromb. Basc.
Biol. 27: 15721579.
3. O, E. et al. (2011) Efficient nonadhesive ex vivo expansion of early endothelial
progenitor cells derived from CD34+ human cord blood fraction for effective
therapeutic vascularization. FASEB J. 25: 159169.
4. Wang, Z. Z. et al. (2007) Endothelial cells derived from human embryonic stem
cells form durable blood vessels in vivo. Nat. Biotechnol. 25: 317318.
5. Bonanno, G. et al. (2009) Interleukin-21 induces the differentiation of human
umbilical cord blood CD34-lineage cells into pseudomature lytic NK cells.
BMC Immunol. 10: 46.

All protocols and data sheets are available at www.miltenyibiotec.com.

140-001-950.05

Unless otherwise specifically indicated, Miltenyi Biotec

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

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