Multicolor flow cytometry analysis of human pluripotent stem cell cultures Background
Antibody
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.
Order no.
Anti-TRA-1-60-PE, human
REA157
130-100-347
Anti-SSEA-1-PE-Vio 770, human and mouse
REA321
130-104-938
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
130-104-940
Anti-Oct3/4 Isoform A-APC, human and mouse
REA338
130-105-555
Mouse IgG1-VioBlue
IS5-21F5
130-094-670
REA Control (I)-FITC
REA293
130-104-611
REA Control (I)-APC
REA293
130-104-615
REA Control (S)-PE
REA293
130-104-612
REA Control (S)-VioGreen
REA293
130-104-608
REA Control (S)-PE-Vio 770
REA293
130-104-616
Detection antibodies
Isotype control antibodies
Material
Table 1: Multicolor panel and isotype controls for flow cytometry of PSCs and differentiated precursor cells.
Antibodies Table 1 specifies the antibodies used for the multicolor panel and the corresponding isotype control antibodies. All antibodies are diluted 1:11 for flow cytometry.
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.
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-REA (#130-104-693) to set up the PE-Vio® 770 channel for detection of the differentiation marker SSEA-1
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Clone
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Flow cytometry
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 90 μL of Inside Perm. 14. Resuspend samples 2–7 in 100 μL of Inside Perm. 15. Add 10 µL of each of the following antibodies, detecting intracellular markers, to sample 1: i) Anti-Sox2-FITC, and ii) Anti-Oct3/4 Isoform A-APC. 16. Add 10 µL of one of the antibodies for intracellular markers to samples 6 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.
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-REA 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 70 μL of buffer. This will be the cell sample stained with the complete panel. 4. Resuspend samples 2–7 in 100 µL of buffer. This 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-TRA-1-60-PE, ii) Anti-SSEA-1-PE-Vio 770, iii) Anti-SSEA-4-VioGreen™, and iv) Anti-SSEA-5-VioBlue®. 6. Add 10 µL of one of the antibodies detecting a surface marker to samples 3–5. 7. Do not add antibody to samples 2, 6, and 7 at this point. Cell surface staining
Intracellular staining
Buffer
Anti-TRA1-60-PE
Anti-SSEA-1PE-Vio 770
Anti-SSEA-4VioGreen
Anti-SSEA-5VioBlue
1. Multicolor panel
70 µL
10 µL
10 µL
10 µL
2. Unstained sample
100 µL
–
–
–
Sample
Inside Perm
Anti-Sox2FITC
Anti-Oct3/4 Isoform A-APC
10 µL
90 µL
10 µL
10 µL
–
100 µL
–
–
3. Single-color staining
100 µL
10 µL
–
–
–
100 µL
–
–
4. Single-color staining
100 µL
–
–
10 µL
–
100 µL
–
–
5. Single-color staining
100 µL
–
–
–
10 µL
100 µL
–
–
6. Single-color staining
100 µL
–
–
–
–
100 µL
10 µL
–
7. Single-color staining
100 µL
–
–
–
–
100 µL
–
10 µL
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.
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Isotype controls Isotype 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.
A
-1 0 1
250
500
Forward scatter
B
Anti-TRA-1-60-PE
10² 10¹ 1 0 0.45% -1 -1 0 1
0.00%
10¹
10²
10²
10³
Compensated
10³
21.27%
78.28%
0.18%
10¹ FITC-A
Uncompensated
10³
10³
99.23%
-1 0 1
0.29%
10² 10¹ 1 0 0.47% -1 -1 0 1
0.00%
10¹
10²
10³
FITC-A
C
10¹
-1 0 1
PE-A
250
10²
10³
10¹
10²
10³
REA Control (S)-VioGreen
10²
10³
-1 0 1
10¹
10²
10³
REA Control (I)-APC
-1 0 1
10¹
10²
10³
Mouse IgG1-VioBlue
0
250
500
750 1000
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.
10¹ 1 0 -1 -1 0 1
10¹
10²
Forward scatter
PE-Vio 770-A
Uncompensated
Compensated
10³
10³
10²
10² PE-A
Side scatter
10¹
REA Control (S)-PE
10²
500
0
PE-A
10³
FSC/SSC 99.92%
750
-1 0 1
REA Control (S)-PE-Vio 770
FITC-A
1000
10³
10¹ 1 0 98.87% -1 -1 0 1
750 1000
10²
Relative cell number
0
10¹
REA Control (I)-FITC
Relative cell number
PE-A
250
Anti-TRA-1-60-PE
0.28%
10²
500
0
0.67%
Relative cell number
750 Side scatter
10³
FSC/SSC 64.91%
Relative cell number
1000
Relative cell number
Relative cell number
Instrument setup 1. 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.
10¹ 1 0 -1 -1 0 1
10¹
10²
10³
Anti-SSEA-1-PE-Vio 770
Results
10³
The 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, Sox-2, and Oct 3/4 were expressed at high levels, whereas expression of the differentiation marker SSEA-1 was low.
10¹ 1 0 -1 -1 0 1
10¹
10²
10³
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 (fig. 3B). Sox2 was expressed at high levels, as could be expected from neural precursors (fig. 3B).
Anti-SSEA-1-PE-Vio 770
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-REA according to the accompanying protocol. PE-Vio 770 vs. PE is shown as an example.
Multicolor flow cytometry analysis of hPSCs | February 2016
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).
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10³
10² 10¹ 1 0 0.19% -1 -1 0 1
0.27%
10¹
10²
10³
0.18%
10² 10¹ 1 0 0.24% -1 -1 0 1
10³
0.85%
10³
98.58%
10² 10¹ 1 0 0.38% -1 -1 0 1
0.19%
10¹
10²
0.34%
10¹
10²
10³
0.30%
10³
0.40%
10¹ 1 0 0.21% -1 -1 0 1
98.99%
10¹
Anti-Sox2-FITC
0.34%
10¹
10²
1.50%
10¹
10²
10³
10³
10³
10³
0.03%
0.30%
99.16%
10² 10¹ 1 0 0.24% -1 -1 0 1
Anti-TRA-1-60-PE
10²
1 0 0.27% -1 -1 0 1
98.00%
10²
Anti-TRA-1-60-PE
Anti-SSEA-4-VioGreen
Anti-Oct3/4 Isoform A-APC
Anti-TRA-1-60-PE
10³
99.24%
Anti-Sox2-FITC
99.17%
Anti-SSEA-1-PE-Vio 770
0.37%
Anti-SSEA-5-VioBlue
Anti-SSEA-4-VioGreen
10³
Anti-Oct3/4 Isoform A-APC
A
0.29%
10¹
10²
10³
Anti-TRA-1-60-PE
15.19%
10² 10¹ 1 0 0.77% -1 -1 0 1
Anti-SSEA-5-VioBlue
84.02%
10¹
10²
10³
Anti-TRA-1-60-PE
SSEA-1
SSEA-5
SSEA-4
Oct 3/4
7.95
98.74
98.89
Sox2
7.74
97.18
97.23
99.58
TRA-1-60
15.19
99.24
99.17
SSEA-4
12.39
98.99
99.54
SSEA-5
9.90
99.42
SSEA-1
15.23
TRA-1-60
Sox2
Oct 3/4
99.16
98.58
99.46
98.00
98.77
10¹ 1 0 66.24% -1 -1 0 1
0.50%
10¹
10²
10³
10² 10¹ 1 0 98.58% -1 -1 0 1
0.37%
10³
5.53%
10² 10¹ 1 0 2.49% -1 -1 0 1
91.61%
10¹
10²
0.69%
10¹
10²
10³
96.88%
10³
31.02%
10¹ 1 0 2.51% -1 -1 0 1
0.55%
10¹
Anti-Sox2-FITC
0.11%
10¹
10²
0.07%
10¹
10²
10³
10³
10³
10³
40.35%
10¹ 1 0 92.74% -1 -1 0 1
10¹
Anti-SSEA-5-VioBlue
10³
0.49%
0.31%
10¹
10²
0.27%
0.39%
10¹
10²
10³
Anti-TRA-1-60-PE
10²
1 0 58.85% -1 -1 0 1
6.60%
10²
Anti-TRA-1-60-PE
10²
1 0 68.32% -1 -1 0 1
0.54%
10²
Anti-TRA-1-60-PE
Anti-SSEA-4-VioGreen
Anti-Oct3/4 Isoform A-APC
Anti-TRA-1-60-PE 10³
10³
0.22%
Anti-Sox2-FITC
10²
0.51%
Anti-Pax6-PE
10³
0.80%
Anti-SSEA-1-PE-Vio 770
32.46%
Anti-SSEA-5-VioBlue
Anti-SSEA-4-VioGreen
10³
Anti-Oct3/4 Isoform A-APC
B
10³
Anti-TRA-1-60-PE
8.94%
79.65%
10² 10¹ 1 0 5.81% -1 -1 0 1
5.60%
10¹
10²
10³
Anti-PSA-NCAM-APC
SSEA-1
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
43.96
low
marker expression
high
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 (bold numbers) and double-positive cells. Multicolor flow cytometry analysis of hPSCs | February 2016
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Conclusion
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). A
The 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.
B
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.
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Multicolor flow cytometry analysis of hPSCs | February 2016
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