1 Background Thorough characterization of pluripotency is mandatory during the establishment of newly derived human embryonic or induced pluripotent stem cell (hiPSC) lines. However, it is equally important to constantly monitor established cell lines in order to detect signs of spontaneous differentiation early on. Monitoring of the pluripotency and differentiation status of PSC cultures is commonly done by immunofluorescence microscopy analysis. This method, however, is laborious and does not allow for reliable quantification of cell populations. Despite its vast potential for sophisticated and detailed cell analysis, flow cytometry is used only seldom in stem cell research. Here we present a multicolor flow cytometry protocol that allows for the simultaneous quantification of intracellular and surface markers for easy monitoring of the pluripotency status of human PSC cultures. Material Antibodies Table 1 specifies the antibodies used for the multicolor panel and the corresponding isotype control antibodies. The recommended dilution for each antibody is specified in the datasheet. Buffers and reagents • Buffer consisting of PBS, pH 7.2, 0.5% BSA, 2 mM EDTA is prepared by diluting MACS® BSA Stock Solution (#130-091-376) 1:20 with autoMACS® Rinsing Solution (#130-091-222). Keep the buffer cold (2−8 °C). • Inside Stain Kit (#130-090-477) for intracellular staining. • MACS Comp Bead Kit, anti-mouse Igκ (#130-097-900) to set up the PE-Vio® 770 channel for detection of the differentiation marker SSEA-1 (CD15). Multicolor flow cytometry analysis of human pluripotent stem cell cultures Antibody Clone Order no. Detection antibodies Anti-TRA-1-60-PE, human REA157 130-122-921 CD15-PE-Vio770, human VIMC6 130-113-486 Anti-SSEA-4-VioGreen, human REA101 130-098-341 Anti-SSEA-5-VioBlue, human 8e11 130-106-657 Anti-Sox2-FITC, human and mouse REA320 130-120-721 Anti-Oct3/4 Isoform A-APC, human and mouse REA338 130-117-709 Isotype control antibodies Mouse IgG1-VioBlue IS5-21F5 130-113-205 REA Control (I)-FITC REA293 130-118-354 REA Control (I)-APC REA293 130-120-709 REA Control (S)-PE REA293 130-113-438 REA Control (S)-VioGreen REA293 130-113-444 Table 1: Multicolor panel and isotype controls for flow cytometry of PSCs and differentiated precursor cells. Cells Human PSCs were cultured in StemMACS™ iPS-Brew XF, human (#130-104-368), in 6-well plates coated with Matrigel® hESC-Qualified Matrix. To confirm that the flow cytometry assay reliably detects a loss of pluripotency, we also analyzed hiPSCs differentiated towards the neural lineage. Both pluripotent and differentiated cells were treated with 0.05% trypsin/EDTA to obtain single cells for flow cytometry analysis.
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Multicolor flow cytometry analysis of human pluripotent ... · Figure 3: Multicolor flow cytometry analysis of undifferentiated (A) and differentiated (B) human iPSCs. Cells were
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1
BackgroundThorough characterization of pluripotency is mandatory during the establishment of newly derived human embryonic or induced pluripotent stem cell (hiPSC) lines. However, it is equally important to constantly monitor established cell lines in order to detect signs of spontaneous differentiation early on. Monitoring of the pluripotency and differentiation status of PSC cultures is commonly done by immunofluorescence microscopy analysis. This method, however, is laborious and does not allow for reliable quantification of cell populations. Despite its vast potential for sophisticated and detailed cell analysis, flow cytometry is used only seldom in stem cell research. Here we present a multicolor flow cytometry protocol that allows for the simultaneous quantification of intracellular and surface markers for easy monitoring of the pluripotency status of human PSC cultures.
MaterialAntibodiesTable 1 specifies the antibodies used for the multicolor panel and the corresponding isotype control antibodies. The recommended dilution for each antibody is specified in the datasheet.
Buffers and reagents• Buffer consisting of PBS, pH 7.2, 0.5% BSA, 2 mM EDTA
is prepared by diluting MACS® BSA Stock Solution (#130-091-376) 1:20 with autoMACS® Rinsing Solution (#130-091-222). Keep the buffer cold (2−8 °C).
• Inside Stain Kit (#130-090-477) for intracellular staining. • MACS Comp Bead Kit, anti-mouse Igκ (#130-097-900) to
set up the PE-Vio® 770 channel for detection of the differentiation marker SSEA-1 (CD15).
Multicolor flow cytometry analysis of human pluripotent stem cell cultures
Antibody Clone Order no.
Detection antibodies
Anti-TRA-1-60-PE, human REA157 130-122-921
CD15-PE-Vio770, human VIMC6 130-113-486
Anti-SSEA-4-VioGreen, human REA101 130-098-341
Anti-SSEA-5-VioBlue, human 8e11 130-106-657
Anti-Sox2-FITC, human and mouse REA320 130-120-721
Anti-Oct3/4 Isoform A-APC, human and mouse
REA338 130-117-709
Isotype control antibodies
Mouse IgG1-VioBlue IS5-21F5 130-113-205
REA Control (I)-FITC REA293 130-118-354
REA Control (I)-APC REA293 130-120-709
REA Control (S)-PE REA293 130-113-438
REA Control (S)-VioGreen REA293 130-113-444
Table 1: Multicolor panel and isotype controls for flow cytometry of PSCs and differentiated precursor cells.
CellsHuman PSCs were cultured in StemMACS™ iPS-Brew XF, human (#130-104-368), in 6-well plates coated with Matrigel® hESC-Qualified Matrix. To confirm that the flow cytometry assay reliably detects a loss of pluripotency, we also analyzed hiPSCs differentiated towards the neural lineage. Both pluripotent and differentiated cells were treated with 0.05% trypsin/EDTA to obtain single cells for flow cytometry analysis.
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Flow cytometry Cell staining The panel for analysis of PSCs includes antibodies for both surface markers and intracellular markers. Surface markers are stained first. Subsequently, cells are fixed and permeabilized for intracellular staining.
To set up the instrument and compensate for spectral overlap, single stainings for each antibody and an unstained cell sample are required. The unstained sample does not contain any antibody, but is otherwise treated, e.g., fixed and permeabilized, in the same way as the stained samples. Please see the pipetting scheme in table 2 for an overview. As SSEA-1 is not expressed in pluripotent cells, the MACS® Comp Bead Kit, anti-mouse Igκ is used for compensation of PE-Vio 770 instead of a cell sample. Note: Fluorescence-minus-one (FMO) controls might help you to set the gates more accurately. FMO controls contain all antibodies except for one.
1. Determine cell number. Use 1×10⁶ iPS cells per sample.2. Centrifuge cell suspension at 300×g for 5 minutes.
Aspirate supernatant completely.3. Resuspend sample 1 in 85.6 μL of buffer. This will be the
cell sample stained with the complete panel.4. Resuspend sample 3 in 107.8 µL buffer. Resuspend
samples 2, 4, 5, 6, and 7 in 100 µL buffer. These will be the unstained and single-stained samples.
5. Add 10 µL of each of the following antibodies, detecting the surface markers, to sample 1: i) Anti-SSEA-4-VioGreen™ and ii) Anti-SSEA-5-VioBlue®. Add 2.2 µL of CD15-PE-Vio770 and Anti-TRA-1-60-PE.
6. Add 2.2 uL of Anti-TRA-1-60-PE to sample 3. Add 10 µL of Anti-SSEA-4-VioGreen and Anti-SSEA-5-VioBlue to samples 4 and 5, respectively.
7. Do not add antibody to samples 2, 6, and 7 at this point.8. Mix well and incubate for 10 minutes in the dark in the
refrigerator (2−8 °C). Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
9. Wash cells by adding 1 mL of buffer per sample and centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
10. Resuspend all samples in 100 µL of buffer. Add 100 µL of Inside Fix per sample.
11. Mix well and incubate for 20 minutes in the dark at room temperature.
12. Wash cells by adding 1 mL of buffer and centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
13. Resuspend sample 1 in 105.6 μL of Inside Perm.14. Resuspend samples 2–5 in 100 μL of Inside Perm.
Resuspend samples 6 and 7 in 107.8 μL of Inside Perm.15. Add 2.2 µL of Anti-Sox2-FITC, to sample 1 and 6.16. Add 2.2 µL of Anti-Oct3/4 Isoform A-APC to samples
1 and 7.17. Mix well and incubate for 15 minutes in the dark at room
temperature.18. Wash cells by adding 1 mL of Inside Perm and centrifuge
at 300×g for 5 minutes. Aspirate supernatant completely.
19. Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry. A volume of 1 mL of buffer per sample is recommended. Note: Fixed and permeabilized cells are smaller than viable cells. Thus, FSC/SSC settings of the flow cytometer might have to be adjusted.
Table 2: Pipetting scheme for surface and intracellular staining. Cell surface staining refers to steps 3–8 of the protocol. Intracellular staining refers to steps 14–19.
3
Instrument setup1. Set up the scatter and voltages for all channels with
the unstained cell sample (sample 2) (fig. 1A). Specify the trigger.
2. Use single-color stainings (samples 3–7) to define the compensation (fig. 1B).
3. To set up the PE-Vio 770 channel, temporarily lower the trigger to detect all the Comp Beads and define the compensation (fig. 1C). Afterwards change the trigger settings back to the value specified in step 1.
Figure 1: Setting up the flow cytometer for compensation of spectral overlap. (A) Adjustment of scatter and voltage using an unstained sample. PE vs. FITC is shown as an example. (B) Compensation using a PE-stained cell sample; PE vs. FITC is shown as an example. (C) Compensation of the PE-Vio 770 channel using MACS® Comp Bead Kit, anti-mouse Igκ according to the accompanying protocol. PE-Vio 770 vs. PE is shown as an example.
Isotype controlsIsotype controls are used to check for non-specific binding of the various fluorochrome-conjugated antibodies to cells. Cells are incubated with the isotype control antibodies following the instructions above as indicated for sample 1 and analyzed accordingly. Figure 2 shows a representative example of isotype control staining.
Figure 2: Isotype control staining. Cells were incubated with the various isotype control antibodies (red line) or left unstained (black line) and analyzed by flow cytometry on the MACSQuant® Analyzer 10.
10³-1 10¹ 10²0 1
REA Control (I)-FITC
Re
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ell n
umb
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10³-1 10¹ 10²0 1
REA Control (S)-PE
Re
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umb
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10³-1 10¹ 10²0 1
REA Control (I)-APC
Re
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ell n
umb
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10³-1 10¹ 10²0 1
REA Control (S)-VioGreen
Re
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10³-1 10¹ 10²0 1
Mouse IgG1-VioBlue
Re
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ResultsThe antibody panel described here enables the detection of both surface and intracellular markers for monitoring pluripotency. Figure 3A shows a representative result for the analysis of human iPSCs. The pluripotency markers SSEA-4, SSEA-5, TRA-1-60, Sox2, and Oct 3/4 were expressed at high levels, whereas expression of the differentiation marker SSEA-1 (CD15) was low.
In contrast, cells differentiated towards the neural lineage expressed the pluripotency markers at low levels, or even shut off the expression of pluripotency markers, and up-regulated the differentiation marker SSEA-1 (CD15) (fig. 3B). Sox2 was expressed at high levels, as could be expected from neural precursors (fig. 3B).
As a differentiation control, we stained a second sample with antibodies against PSA-NCAM and Pax6, which are markers for neuronal and neural progenitors, respectively. The large majority of cells (80%) co-expressed both markers (fig. 3B).
A
100000
500 750
250
500
750
1000
250
Forward scatter
Si
de
scat
ter
FSC/SSC64.91%
10³-101
10¹ 10²0
10³
10²
10¹
PE-Vio 770-A
PE-A
-1 1
B
10³-101
10¹ 10²0
10³
10²
10¹
FITC-A
Ant
i-TR
A-1
-60-
PE
-1 1
78.28% 21.27%
0.45% 0.00%
10³-101
10¹ 10²0
10³
10²
10¹
FITC-A
Ant
i-TR
A-1
-60-
PE
-1 1
99.23% 0.29%
0.47% 0.00%
C
100000
500 750
250
500
750
1000
250
Forward scatter
Si
de
scat
ter
FSC/SSC99.92%
10³-101
10¹ 10²0
10³
10²
10¹
FITC-A
PE-A
-1 1
0.67% 0.28%
98.87% 0.18%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-SSEA-1-PE-Vio 770
PE-A
-1 1 10³-101
10¹ 10²0
10³
10²
10¹
Anti-SSEA-1-PE-Vio 770
PE-A
-1 1
Uncompensated Compensated
Uncompensated Compensated
CD15-PE-Vio770 CD15-PE-Vio770
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Figure 3: Multicolor flow cytometry analysis of undifferentiated (A) and differentiated (B) human iPSCs. Cells were stained with the antibodies as indicated and analyzed by flow cytometry on the MACSQuant® Analyzer 10. Unstained cells were used as a control for gating. Numbers in the heat map specify percentages of single-positive and double-positive (bold numbers) cells.
A
SSEA-1 (CD15) SSEA-5 SSEA-4 TRA-1-60 Sox2 Oct 3/4
Oct 3/4 7.95 98.74 98.89 99.16 98.58 99.46
Sox2 7.74 97.18 97.23 98.00 98.77
TRA-1-60 15.19 99.24 99.17 99.58
SSEA-4 12.39 98.99 99.54
SSEA-5 9.90 99.42
SSEA-1 (CD15) 15.23
B
SSEA-1 (CD15) SSEA-5 SSEA-4 TRA-1-60 Sox2 Oct 3/4
Oct 3/4 1.75 0.33 1.80 0.27 5.53 6.87
Sox2 40.51 0.78 27.73 0.54 97.14
TRA-1-60 0.49 0.22 0.80 1.30
SSEA-4 7.04 0.55 33.26
SSEA-5 0.32 0.85
SSEA-1 (CD15) 43.96
low highmarker expression
10³-101
10¹ 10²0
10³
10²
10¹
Anti-TRA-1-60-PE
Ant
i-SS
EA-4
-Vio
Gre
en
-1 1
0.37% 99.17%
0.19% 0.27%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-TRA-1-60-PEA
nti-
SSEA
-5-V
ioB
lue
-1 1
0.18% 99.24%
0.24% 0.34%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-TRA-1-60-PE
Ant
i-So
x2-F
ITC
-1 1
0.30% 98.00%
0.21% 1.50%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-TRA-1-60-PE
Ant
i-O
ct3/
4 Is
ofor
m A
-APC
-1 1
0.30% 99.16%
0.24% 0.29%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-Sox2-FITC
Ant
i-O
ct3/
4 Is
ofor
m A
-APC
-1 1
0.85% 98.58%
0.38% 0.19%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-SSEA-5-VioBlue
Ant
i-SS
EA-4
-Vio
Gre
en
-1 1
0.40% 98.99%
0.27% 0.34%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-TRA-1-60-PEA
nti-
SSEA
-1-P
E-V
io 7
70
-1 1
0.03% 15.19%
0.77% 84.02%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-TRA-1-60-PE
Ant
i-SS
EA-4
-Vio
Gre
en
-1 1
32.46% 0.80%
66.24% 0.50%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-TRA-1-60-PE
Ant
i-SS
EA-5
-Vio
Blu
e
-1 1
0.51% 0.22%
98.58% 0.69%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-TRA-1-60-PE
Ant
i-So
x2-F
ITC
-1 1
96.88% 0.54%
2.51% 0.07%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-TRA-1-60-PE
Ant
i-O
ct3/
4 Is
ofor
m A
-APC
-1 1
6.60% 0.27%
92.74% 0.39%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-Sox2-FITC
Ant
i-O
ct3/
4 Is
ofor
m A
-APC
-1 1
0.37% 5.53%
2.49% 91.61%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-SSEA-5-VioBlue
Ant
i-SS
EA-4
-Vio
Gre
en
-1 1
31.02% 0.55%
68.32% 0.11%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-TRA-1-60-PE
Ant
i-SS
EA-1
-PE-
Vio
770
-1 1
40.35% 0.49%
58.85% 0.31%
10³-101
10¹ 10²0
10³
10²
10¹
Anti-PSA-NCAM-APC
Ant
i-Pa
x6-P
E
-1 1
8.94% 79.65%
5.81% 5.60%
CD15
-PE-Vio77
0CD
15-PE-Vio77
0
PSC cultures were also monitored visually by light microscopy. The images show the typical morphology of PSC colonies (fig. 4A) or cells differentiated into neural precursors (fig. 4B).
ConclusionThe antibody panel described in this application note enables reliable monitoring of the pluripotency status of PSCs by multicolor flow cytometry. The procedure is fast and easy to perform for routine monitoring. Intracellular and surface markers are detected simultaneously.
Figure 4: Cultures of undifferentiated human iPSCs (A) and iPSCs differentiated towards the neural lineage (B). Shown are light microscopy images of the same cultures that were used for flow cytometry.