For research use only
Enumeration of CD34/CD133 positive cells with the CD34/CD133 Enumeration Kit
Applications for the MACSQuant® Analyzer*
Background
Therefore, CD133+ stem cells have come into focus in nonhematological applications especially in ischemic heart
diseases.¹⁹¯²⁶
The CD34 antigen is a single-chain transmembrane
glycoprotein, expressed on human hematopoietic stem
and progenitor cells constituting a small subpopulation of
bone marrow cells and peripheral blood cells. The antigen
is absent on fully differentiated hematopoietic cells, such
as normal peripheral blood lymphocytes, monocytes,
granulocytes, erythrocytes, and platelets. After severe
damage, for example, after myeloablative conditioning,
the hematopoietic system can be reconstituted by
transplantation of allogenic or autologous CD34+
hematopoietic progenitor cells (HPC). In the CD34/CD133
Enumeration Kit the antibody clone AC136 recognizes a
class III epitope of the CD34 antigen.
CD133 is a 5-transmembrane cell surface antigen, expressed
on a subset of CD34 bright stem and progenitor cells in
human fetal liver, bone marrow, cord blood, and peripheral
blood but is not found on mature blood cells.¹ In contrast
to the CD34 antigen, CD133 is not expressed by late
progenitors, such as pre-B-cells, CFU-E, and CFU-G.², ³
CD133 has also been found to be expressed on circulating
endothelial progenitor cells and fetal neural stem cells as
well as on other tissue-specific stem cells, such as renal,
prostate, and corneal stem cells.⁴¯⁶ In the CD34/CD133
Enumeration Kit the CD133/2 (clone 239C3) antibody
recognizes epitope 2 of the CD133 antigen.
CD34+ and CD34+CD133+ cells may have various therapeutic
uses. CD34 and CD133 enriched stem cell grafts have been
employed in autologous⁷¯⁸ and allogenic transplantation
both in the haploidentical⁹¯¹¹ as well as the HLA-matched
setting¹². The high potential for hematopoietic engraftment
of isolated CD133+ cells has been shown in NOD/SCID
repopulation assays.¹³,¹⁴ CD34 and CD133 enriched cell
fractions have also been used as starting fraction for
ex vivo expansion of hematopoietic progenitor cells from
cord blood.¹⁵¯¹⁸
Moreover CD133+ stem cells can be utilized in nonhematological applications, such as regenerative medicine.
These cells have been shown to harbor the capability to
differentiate into cell types of various tissues, for example,
endothelial cells, neural cells, and hepatocytes.
Applications for the MACSQuant® Analyzer | June 2013
CD34+/CD133+ HPC enumeration using
the MACSQuant® Analyzer
The CD34/CD133 HPC Enumeration Kit is designed for the
accurate detection of CD34+/CD133+ HPCs from human
blood samples by flow cytometry. The kit contains all of the
necessary stainings for CD34/CD133 detection as well as
controls for CD133+ HPC enumeration and viability.
The gating strategy of the CD34/CD133 Enumeration
Kit is based on the ISHAGE guidelines²⁷ for simple and
standardized enumeration of CD34+ cells.
By employing anti-CD45-FITC, anti-CD34-APC, and antiCD133/2-PE the kit allows the identification of CD45+
leucocytes, CD34+ HPCs, and CD34/CD133 HPCs. CD34+
as well as CD34+/CD133+ HPCs are characterized by a dim
CD45+ fluorescence and a low side scatter.
The kit is designed for single platform technique²⁸,
enabling absolute cell count determination with the
MACSQuant® Analyzer without the need to add further
reagents, for example, counting beads.
Dead cells can be excluded from the analysis by addition
of the DNA stain 7-aminoactinomycin D (7-AAD), which
is also included in the kit. 7-AAD diffuses through the cell
membrane of dead cells and intercalates with their DNA.
Materials and Methods
Reagents and Solutions
• CD34/CD133 Enumeration Kit
(# 170-070-709, for in vitro diagnostic use)
• Cell sample
• Double-distilled water
* Applications on the MACSQuant Analyzer, MACSQuant Analyzer 10,
and MACSQuant VYB are for research use only.
1
Copyright © 2013 Miltenyi Biotec GmbH. All rights reserved.
• B
uffer for optional dilution steps: Prepare a PEB (PBS/
EDTA/BSA) buffer containing phosphate-buffered saline
(PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and
2 mM EDTA, e.g., by diluting MACS® BSA Stock Solution
(# 130-091-376) 1:20 with autoMACS® Rinsing Solution
(# 130-091-222). Keep buffer cold (2−8 °C).
Flow cytometric data acquisition and analysis with
the MACSQuant Analyzer
Note: For detailed information on operating the
MACSQuant Instrument, please refer to the MACSQuant
Instrument user manual and the MACSQuantifyTM
Software guide.
Note: For one test at least 200 µL of cell sample are required
(analysis sample and control sample). We recommend to
stain the analysis sample in dublicates to gain statistically
relevant results.
1.The analysis is performed with the single tube holder.
Make sure the single tube holder is attached to the
instrument and select single tube holder from the
Rack drop down menu.
2.Load the appropriate instrument settings for the cell
type by clicking Open, Instrument Settings, and select
the correct instrument settings with compensation.
3.Open two analysis windows, the first one containing
nine dot plots and the second one containing at least
one large statistics display.
Define the plot windows as follows:
A Plot: Forward scatter versus side scatter
B Plot: CD45-FITC versus side scatter
C Plot: 7-AAD versus side scatter
D Plot: CD34-APC versus side scatter
E Plot: CD45-FITC versus side scatter
F Plot: Forward scatter versus side scatter
G Plot: CD133/2-PE versus CD34-APC
H Plot: CD45-FITC versus side scatter
I Plot: Forward scatter versus side scatter
4.Set the trigger on the CD45-FITC channel (B1) during
data acquisition. Choose Edit, Instrument Settings,
and select the trigger on the FITC-Channel (B1).
Materials
• MACSQuant Analyzer
• Micropipettes with disposable tips: variable
micropipettes with volume ranges of 10–100 μL
and 100–1000 μL
• Vortex mixer
• Disposable capped polystyrene tubes 12.75 mm
for use whith MACSQuant Analyzer
Sample preparation
The following protocol can be used for CD34/CD133 HPC
enumeration from various human blood products, such
as whole blood, leukapheresis harvest, bone marrow, cord
blood, and all fractions of CD34 + and CD133+ cells separated
using a CliniMACS® System.
Staining of CD34+/CD133+ cells
Please refer to the package insert of the CD34/CD133
Enumeration Kit for sample requirements and preparation
of solutions.
1.For each sample label two 12.75 mm tubes with A
(CD133 Control) and B (CD34/CD133 Staining Cocktail).
2.Add 20 μL of CD133 Control into tube A and 20 μL of
CD34/CD133 Staining Cocktail into tube B.
3.Add 20 μL of 7-AAD solution into both tubes.
4.Carefully pipette 100 μL of well-mixed cell sample to the
bottom of each labelled tube. Use reverse pipetting for
all pipetting steps with blood products or RBC Lysis
Solution (see package insert of the CD34/CD133
Enumeration Kit chapter 6). Immediately vortex
thoroughly for 3 seconds and incubate for 10 minutes
at 2–8°C in the dark.
5. Add 1860 μL of 1× RBC Lysis Solution to each tube.
Immediately vortex thoroughly for 3 seconds and
incubate for 10 minutes at room temperature in
the dark.
6.The analysis should be performed within one hour
after staining.
7.Store the samples on ice until analysis.
Note: CD34+ and CD34+/CD133+ progenitor cells express
CD45 with lower staining intensity than lymphocytes.
Be careful when setting the threshold and do not exclude
these events from the analysis.
Figure 1: Setting the threshold on the CD45-FITC channel by selecting
the trigger on the FITC-channel B1.
Staining summary
Sample
type
CD 133
Control [μL]
CD34/133
Staining
Cocktail [μL]
7-AAD
[μL]
Specimen
[μL]
A: CD133
control
20
–
20
100
B: CD34/
CD133
Staining
cocktail
–
20
20
100
Applications for the MACSQuant® Analyzer | June 2013
2
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Gating overview
5.Acquire samples with height parameter activated which
can be chosen in the instrument settings. Press Edit,
Instrument Settings, Advanced, and mark Height.
Gate
Label
Definition
P2
WBCs
Not P1/P2
P3
Viable WBCs
Not P1/P2/P3
P1
Note: It is important to gate the CD45-FITC staining in
Height (CD45-FITC-H). It does not matter whether the other
channels are gated on Height (...-H) or Area (...-A).
Not P1
P4
Not P1/P2/P3/P4
P5
Cluster control
CD34
Not P1/P2/P3/P4/P5
P6
CD34+ HPCs
Not P1/P2/P3/P4/P5/P6
P8
Cluster control
CD133
Not P1/P2/P3/P4/P5/P6/P7/P8
P9
CD133+ HPCs
Not P1/P2/P3/P4/P5/P6/P7/P8/P9
P7
Not P1/P2/P3/P4/P5/P6/P7
A Plot: Forward scatter (FSC) versus side scatter (SSC)
Activated gate: no gate
Define region P1 in this plot. P1 includes the SSC low and
FSC low events. The events inside this region should be
excluded from the further analysis by defining region P1
as an exclusion gate (“not-region”).
1. Choose the Samples tab
2.Mark the relevant file and open it by clicking on +
3.Choose region P1 and click right on it. By pressing
Region Properties and marking Not the region
becomes a “not-Region”
Figure 2: The parameter Height is activated in the instrument settings.
Note: At least 100.000 CD45 positive events per tube and a
minimum of 100 CD133 positive events should be counted if
possible. When analyzing a target cell fraction of a
CliniMACS® Separation at least 10,000 CD45 positive events
should be counted.
Note: All samples should be acquired in “high”-flowrate if
possible.
Data acquisition and analysis
Analyze the samples in the following order:
1. CD133 control (tube A)
2. CD34/CD133 Staining cocktail (tube B)
Recommended acquisition volumes for different sample
types on MACSQuant Analyzer:
Sample
Recommended
aquisition volumes
Whole blood
Figure 3: Defining P1 as a “not-Region”.
450 μL
Leukapheresis harvest
200–450 μL
Bone marrow
200–450 μL
300–450 μL
CliniMACS CD34 target cell fraction
300–450 μL
CliniMACS CD133+ target cell fraction
300–450 μL
+
Side scatter
Cord blood
Description of the detailed gating strategy
The detailed description of the gating strategy is shown
using a cord blood sample.
Forward scatter
The regions P1–P7 in plot A to G are defined during
acquisition of the CD133 control sample. The regions P8
and P9 in plot H and I are defined during acquisition of the
enumeration sample.
Applications for the MACSQuant® Analyzer | June 2013
B Plot: CD45-FITC-H versus side scatter
Activated gate: not P1
Set P2 to exclude all CD45 negative events from the analysis
while all CD45 positive cells are included.
Note: CD34+ and CD34+/CD133+ progenitor cells express
CD45 with lower staining intensity than lymphocytes.
3
Copyright © 2013 Miltenyi Biotec GmbH. All rights reserved.
F Plot: CD34+ HPC determination plot;
forward scatter versus side scatter
Activated gate: not P1/P2/P3/P4/P5
Define a region P6 that identifies a cluster of events
meeting all the fluorescence and light scatter criteria of
CD34 positive progenitor cells. Cells clustered in region P6
exhibit slightly higher forward scatter than that of small
lymphocytes and uniformly low side scatter. Any events
outside region P6 are not included in the % viable CD34
positive cells determination. The proper setting of this
region should be checked by showing this region in the first
dot plot (A). The region has to enclose all lymphocytes.
Be careful when setting region P2 and do not exclude these
events from the analysis.
Side scatter
1000
750
500
250
0
-1 0 1
10¹
10²
10³
CD45-FITC
Side scatter
C Plot: 7-AAD versus side scatter
Activated gate: not P1/P2 = WBCs
Define P3 to include all 7-AAD negative viable WBCs.
Side scatter
1000
750
500
Forward scatter
250
0
-1 0 1
10¹
10²
G Plot: CD133/2-PE versus CD34-APC
Activated gate: not P1/P2/P3/P4/P5/P6
Define P7 with the CD133 Control (A) to exclude all CD133
negative events. It is important to set region P7 as close as
possible to the CD133 negative events to include all CD133
positive and CD133dim-positive cells.
A maximum of 10 events is allowed in region P7 on the
CD133 control sample.
10³
7-AAD
D Plot: CD34-APC versus side scatter
Activated gate: not P1/P2/P3 = viable WBC
Define P4 to include all CD34 positive cells with a low SSC.
750
CD34/CD133
Staining cocktail
CD133 Control
10³
10³
250
10²
10²
0
-1 0 1
10¹
10²
10³
CD34-APC
CD34-APC
500
CD34-APC
Side scatter
1000
10¹
1
0
-1
-1 0 1
E Plot: CD34 cluster control plot:
CD45-FITC-H versus side scatter
Activated gate: not P1/P2/P3/P4
The CD34+ cells form a cluster with low SSC and dim CD45
fluorescence and are gated in region P5. Non-specifically
stained events are excluded from this region.
10¹
10²
CD133/2-PE
10³
10¹
1
0
-1
-1 0 1
10¹
10²
10³
CD133/2-PE
After defining plots A to G with tube A, apply all gates
to tube B, by activating the A button and using the
next sample button to load the data from tube B.
For more information, please refer to the MACSQuantify
Software guide.
Side scatter
1000
750
500
250
0
-1 0 1
10¹
10²
10³
CD45-FITC
Applications for the MACSQuant® Analyzer | June 2013
4
Copyright © 2013 Miltenyi Biotec GmbH. All rights reserved.
H Plot: CD133+ cluster control plot:
CD45-FITC-H versus side scatter
Activated gate: not P1/P2/P3/P4/P5/P6/P7
The CD34+/CD133+ cells form a cluster with low SSC and
dim CD45 fluorescence and are gated in region P8. Nonspecifically stained events are excluded from this region.
I Plot: CD133+ HPC determination plot;
forward scatter versus side scatter
Activated gate: not P1/P2/P3/P4/P5/P6/P7/P8
Define a region P9 that identifies a cluster of events
meeting all the fluorescence and light scatter criteria of
CD34+/CD133+ progenitor cells. Cells clustered in region P9
exhibit slightly higher forward scatter than that of small
lymphocytes and uniformly low side scatter. Any events
falling outside region P9 are not included in the % viable
CD34+/CD133+ cells determination. The proper setting of
this region should be checked by showing this region in the
first dot plot (A). The region has to enclose all lymphocytes.
Side scatter
1000
750
500
250
0
-1 0 1
10¹
10²
10³
Side scatter
CD45-FITC
Forward scatter
1000
1000
750
750
Side scatter
Side scatter
Sidescatter
scatter
Side
Overview of the complete analyis window containing the dot plots
500
250
0
-1 0 1
Forward scatter
Forward
scatter
10¹
10²
10³
500
250
0
-1 0 1
CD45-FITC
750
500
250
10¹
10²
500
250
0
-1 0 1
10³
1000
10²
750
Side scatter
CD34-APC
10³
10¹
10¹
10²
10¹
10²
10³
Forward scatter
Forward
scatter
CD45-FITC
CD34-APC
1
0
-1
-1 0 1
10³
10³
CD133/2-PE
Applications for the MACSQuant® Analyzer | June 2013
Side
Sidescatter
scatter
0
-1 0 1
10²
Side
Sidescatter
scatter
750
Side scatter
1000
Side scatter
1000
10¹
7-AAD
500
250
0
-1 0 1
10¹
10²
10³
Forward scatter
Forward
scatter
CD45-FITC
5
Copyright © 2013 Miltenyi Biotec GmbH. All rights reserved.
Region
%-#
Count
Count/ml
9.95e5
100.00
199193
P1
77.04
153458
7.67e5
P2
99.11
152091
7.60e5
P3
(A)
97.97
P4
(E)
0.33
P5
88.19
P6
100.00
P7
84.53
P8
99.73
P9
100.00
Analysis of results using the MACSQuant Analyzer
For analysis of results display a statistic table on page two
of the analysis with the following parameters: count,
count/mL, and percentage of superior gate (%#).
The highlighted values are required for calculations.
149011
(B)
491
433
(D)
433
2.16e3
(C)
366
365
2.16e3
1.83e3
365
(G)
7.44e5
2.45e3
1.82e3
(F)
1.82e3
Example of a calculation according to the statistic
table (values of actual cord blood samples shown
in dot plots A-I)
Dilution Factor = 1×2000 μL/100 μL = 20
A: Viability of WBC = 97.97 %
B: Viable WBC [count/mL] = 7.44×10⁵×20
(dilution factor) = 1.49×10⁷
C: Viable CD34+ HPC [count/mL] = 2.16×10³×20
(dilution factor) = 4.32×10⁴
D+E: % viable CD34+ HPC = 433×100/149011 = 0.29 %
F: Viable CD34+/CD133+ HPC count/mL = 1.82×10³×20
(dilution factor) = 3.64×10⁴
G: % viableCD34+/CD133+ HPC = 365×100/149011
= 0.24 %
The dilution factor is calculated by multiplying an optional
pre-dilution factor of the sample (no pre-dilution: factor = 1)
by 2000 μL divided by the sample volume used for staining
(100 μL). Dilution factor in standard application without
predilution = 20.
Dilution factor = Pre-dilution factor×2000 μL/sample volume
A: The viability of WBC can be adopted from the statistic
from P3 %#.
B: The viable WBC [cells/mL] can be derived by multiplying
the table item count/mL P3 with the dilution factor (20).
Viable WBC [cells/mL] = Count/mL P3×Dilution factor
C: The viable CD34+ HPC [cells/mL] can be derived by
multiplying the table item count/mL P6 with the dilution
factor (20).
Viable CD34 + HPC [cells/mL] = Count/mL P6×Dilution factor
D+E: The percentage of viable CD34+ HPC can be calculated
by multiplying the table item count P6 (D) by 100 and
dividing this by the table item Count P3 (E).
% viable CD34 + HPC =
Count P6×100
Count P3
F: The viable CD34+/CD133+ HPC [cells/mL] can be derived
by multiplying the table item count/mL P9 with the
dilution factor (20).
Viable CD34 +/CD133+ HPC [cells/mL] = Count/mL P9×Dilution factor
G: The percentage of viable CD34+/CD133+ HPC can be
calculated by multiplying the table item count P9 (G) by
100 and dividing this by the table item Count P3 (E).
% viable CD34 +/CD133+ HPC =
Count P9×100
Count P3
Applications for the MACSQuant® Analyzer | June 2013
6
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References
1.Yin, A. H. et al. (1997) AC133, a Novel Marker for Human
Hematopoietic Stem and Progenitor Cells. Blood 90: 5002–5012.
2.Freund, D. et al. (2006) Comparative analysis of proliferative potential
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CD133+ blood stem cells derived from a single donor.
Cell Prolif. 39: 325–332.
3.Takahiro, S., et al. (2006) Highly Efficient Ex Vivo Expansion of Human
Hematopoietic Stem Cells Using Delta1-Fc Chimeric Protein.
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mismatched peripheral blood stem cells using CD133+-purified stem
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allogeneic transplantation of CD133+ selected hematopoietic
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CliniMACS, MACS and MACSQuant are a registered trademarks of Miltenyi Biotec GmbH. Copyright © 2013 Miltenyi Biotec GmbH. All rights reserved.
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