Multicolor Flow Cytometry Analysis of Human Pluripotent Stem Cell Cultures

Multicolor flow cytometry analysis of human pluripotent stem cell cultures

Background Antibody Clone Order no. Thorough characterization of pluripotency is mandatory Detection antibodies during the establishment of newly derived human Anti-TRA-1-60-PE, human REA157 130-122-921 embryonic or induced pluripotent stem cell (hiPSC) lines. CD15-PE-Vio770, human VIMC6 130-113-486 However, it is equally important to constantly monitor Anti-SSEA-4-VioGreen, human REA101 130-098-341 established cell lines in order to detect signs of spontaneous differentiation early on. Monitoring of the pluripotency Anti-SSEA-5-VioBlue, human 8e11 130-106-657 and differentiation status of PSC cultures is commonly Anti-Sox2-FITC, human and mouse REA320 130-120-721 done by immunofluorescence microscopy analysis. Anti-Oct3/4 Isoform A-APC, REA338 130-117-709 This method, however, is laborious and does not allow for human and mouse reliable quantification of cell populations. Despite its vast Isotype control antibodies potential for sophisticated and detailed cell analysis, flow Mouse IgG1-VioBlue IS5-21F5 130-113-205 cytometry is used only seldom in stem cell research. Here REA Control (I)-FITC REA293 130-118-354 we present a multicolor flow cytometry protocol that allows for the simultaneous quantification of intracellular and REA Control (I)-APC REA293 130-120-709 surface markers for easy monitoring of the pluripotency REA Control (S)-PE REA293 130-113-438 status of human PSC cultures. REA Control (S)-VioGreen REA293 130-113-444

Table 1: Multicolor panel and isotype controls for flow cytometry Material of PSCs and differentiated precursor cells. Antibodies Cells Table 1 specifies the antibodies used for the multicolor Human PSCs were cultured in StemMACS™ iPS-Brew XF, panel and the corresponding isotype control antibodies. human (#130-104-368), in 6-well plates coated with The recommended dilution for each antibody is specified Matrigel® hESC-Qualified Matrix. To confirm that the flow in the datasheet. cytometry assay reliably detects a loss of pluripotency, we also analyzed hiPSCs differentiated towards the neural Buffers and reagents lineage. Both pluripotent and differentiated cells were • Buffer consisting of PBS, pH 7.2, 0.5% BSA, 2 mM EDTA treated with 0.05% trypsin/EDTA to obtain single cells for is prepared by diluting MACS® BSA Stock Solution flow cytometry analysis. (#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).

1 Flow cytometry 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 Cell staining refrigerator (2−8 °C). The panel for analysis of PSCs includes antibodies for both Note: Higher temperatures and/or longer incubation surface markers and intracellular markers. Surface markers times may lead to non-specific cell labeling. Working are stained first. Subsequently, cells are fixed and on ice requires increased incubation times. permeabilized for intracellular staining. 9. Wash cells by adding 1 mL of buffer per sample and To set up the instrument and compensate for spectral centrifuge at 300×g for 5 minutes. Aspirate supernatant overlap, single stainings for each antibody and an unstained completely. cell sample are required. The unstained sample does not 10. Resuspend all samples in 100 µL of buffer. Add 100 µL contain any antibody, but is otherwise treated, e.g., fixed of Inside Fix per sample. and permeabilized, in the same way as the stained samples. 11. Mix well and incubate for 20 minutes in the dark at Please see the pipetting scheme in table 2 for an overview. room temperature. As SSEA-1 is not expressed in pluripotent cells, the 12. Wash cells by adding 1 mL of buffer and centrifuge at MACS® Comp Bead Kit, anti-mouse Igκ is used for 300×g for 5 minutes. Aspirate supernatant completely. compensation of PE-Vio 770 instead of a cell sample. 13. Resuspend sample 1 in 105.6 μL of Inside Perm. Note: Fluorescence-minus-one (FMO) controls might help 14. Resuspend samples 2–5 in 100 μL of Inside Perm. you to set the gates more accurately. FMO controls contain Resuspend samples 6 and 7 in 107.8 μL of Inside Perm. all antibodies except for one. 15. Add 2.2 µL of Anti-Sox2-FITC, to sample 1 and 6. 1. Determine cell number. Use 1×10⁶ iPS cells per sample. 16. Add 2.2 µL of Anti-Oct3/4 Isoform A-APC to samples 2. Centrifuge cell suspension at 300×g for 5 minutes. 1 and 7. Aspirate supernatant completely. 17. Mix well and incubate for 15 minutes in the dark at room 3. Resuspend sample 1 in 85.6 μL of buffer. This will be the temperature. cell sample stained with the complete panel. 18. Wash cells by adding 1 mL of Inside Perm and centrifuge 4. Resuspend sample 3 in 107.8 µL buffer. Resuspend at 300×g for 5 minutes. Aspirate supernatant samples 2, 4, 5, 6, and 7 in 100 µL buffer. These will be completely. the unstained and single-stained samples. 19. Resuspend cell pellet in a suitable amount of buffer for 5. Add 10 µL of each of the following antibodies, detecting analysis by flow cytometry. A volume of 1 mL of buffer the surface markers, to sample 1: i) Anti-SSEA-4- per sample is recommended. VioGreen™ and ii) Anti-SSEA-5-VioBlue®. Add 2.2 µL of Note: Fixed and permeabilized cells are smaller than CD15-PE-Vio770 and Anti-TRA-1-60-PE. viable cells. Thus, FSC/SSC settings of the flow cytometer might have to be adjusted. 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.

Cell surface staining Intracellular staining

Anti-TRA- CD15-PE- Anti-SSEA-4- Anti-SSEA-5- Inside Anti- Anti-Oct3/4 Sample Buffer 1-60-PE Vio770, human VioGreen VioBlue Perm Sox2-FITC Isoform A-APC 1. Multicolor panel 85.6 µL 2.2 µL 2.2 µL 10 µL 10 µL 105.6 µL 2.2 µL 2.2 µL 2. Unstained sample 100 µL – – – – 100 µL – – 3. Single-color staining 107.8 µL 2.2 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 – – – – 107.8 µL 2.2 µL – 7. Single-color staining 100 µL – – – – 107.8 µL – 2.2 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.

2 Instrument setup Isotype controls 1. Set up the scatter and voltages for all channels with Isotype controls are used to check for non-specific binding the unstained cell sample (sample 2) (fig. 1A). Specify of the various fluorochrome-conjugated antibodies to cells. the trigger. Cells are incubated with the isotype control antibodies 2. Use single-color stainings (samples 3–7) to define the following the instructions above as indicated for sample 1 compensation (fig. 1B). and analyzed accordingly. Figure 2 shows a representative 3. To set up the PE-Vio 770 channel, temporarily lower the example of isotype control staining. 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.

1000 10³ FSC/SSC 0.67% 0.28% Relative cell number cell Relative 64.91% number cell Relative 750 10² -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³

500 10¹ REA Control (I)-FITC REA Control (S)-PE PE-A

Side scatter 250 1 0 98.87% 0.18% 0 -1 0 250 500 750 1000 -1 0 1 10¹ 10² 10³

Forward scatter FITC-A B

Uncompensated Compensated number cell Relative Relative cell number cell Relative 10³ 10³ 78.28% 21.27% 99.23% 0.29% -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ 10² 10² REA Control (I)-APC REA Control (S)-VioGreen

10¹ 10¹

1 1 Anti-TRA-1-60-PE Anti-TRA-1-60-PE 0 0 0.45% 0.00% 0.47% 0.00%

-1 -1 -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ FITC-A FITC-A C number cell Relative -1 0 1 10¹ 10² 10³ 1000 FSC/SSC 10³ 99.92% Mouse IgG1-VioBlue 750 10²

500 10¹ Figure 2: Isotype control staining. Cells were incubated with the PE-A various isotype control antibodies (red line) or left unstained (black Side scatter 250 1 line) and analyzed by flow cytometry on the MACSQuant® Analyzer 10. 0 0 -1 0 250 500 750 1000 -1 0 1 10¹ 10² 10³ Forward scatter PE-Vio 770-A Results

Uncompensated Compensated The antibody panel described here enables the detection 10³ 10³ of both surface and intracellular markers for monitoring

10² 10² pluripotency. Figure 3A shows a representative result for the analysis of human iPSCs. The pluripotency markers SSEA-4, 10¹ 10¹ PE-A PE-A SSEA-5, TRA-1-60, Sox2, and Oct 3/4 were expressed at high

1 1 levels, whereas expression of the differentiation marker 0 0 SSEA-1 (CD15) was low. -1 -1 -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ Anti-SSEA-1-PE-VioCD15-PE-Vio770 770 Anti-SSEA-1-PE-VioCD15-PE-Vio770 770 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 Figure 1: Setting up the flow cytometer for compensation of spectral overlap. (A) Adjustment of scatter and voltage using up-regulated the differentiation marker SSEA-1 (CD15) an unstained sample. PE vs. FITC is shown as an example. (fig. 3B). Sox2 was expressed at high levels, as could be (B) Compensation using a PE-stained cell sample; PE vs. FITC is expected from neural precursors (fig. 3B). 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. 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).

3 A 10³ 10³ 10³ 10³ 0.37% 99.17% 0.18% 99.24% 0.30% 98.00% 0.30% 99.16%

10² 10² 10² 10²

10¹ 10¹ 10¹ 10¹

1 1 Anti-Sox2-FITC 1 1

0 Anti-SSEA-5-VioBlue 0 0 0 Anti-SSEA-4-VioGreen 0.19% 0.27% 0.24% 0.34% 0.21% 1.50% 0.24% 0.29%

-1 -1 -1 Anti-Oct3/4 A-APC Isoform -1 -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ Anti-TRA-1-60-PE Anti-TRA-1-60-PE Anti-TRA-1-60-PE Anti-TRA-1-60-PE

10³ 10³ 10³ 0.85% 98.58% 0.40% 98.99% 0.03% 15.19%

10² 10² 10²

10¹ 10¹ 10¹

1 1 CD15-PE-Vio770 1 0 0 0 Anti-SSEA-4-VioGreen

0.38% 0.19% 0.27% 0.34% 770 Anti-SSEA-1-PE-Vio 0.77% 84.02%

Anti-Oct3/4 A-APC Isoform -1 -1 -1 -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ Anti-Sox2-FITC Anti-SSEA-5-VioBlue Anti-TRA-1-60-PE

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 10³ 10³ 10³ 10³ 32.46% 0.80% 0.51% 0.22% 96.88% 0.54% 6.60% 0.27%

10² 10² 10² 10²

10¹ 10¹ 10¹ 10¹

1 1 Anti-Sox2-FITC 1 1

0 Anti-SSEA-5-VioBlue 0 0 0 Anti-SSEA-4-VioGreen 66.24% 0.50% 98.58% 0.69% 2.51% 0.07% 92.74% 0.39%

-1 -1 -1 Anti-Oct3/4 A-APC Isoform -1 -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ Anti-TRA-1-60-PE Anti-TRA-1-60-PE Anti-TRA-1-60-PE Anti-TRA-1-60-PE

10³ 10³ 10³ 10³ 0.37% 5.53% 31.02% 0.55% 40.35% 0.49% 8.94% 79.65%

10² 10² 10² 10²

10¹ 10¹ 10¹ 10¹ Anti-Pax6-PE

1 1 CD15-PE-Vio770 1 1 0 0 0 0 Anti-SSEA-4-VioGreen 5.81% 5.60% 2.49% 91.61% 68.32% 0.11% 770 Anti-SSEA-1-PE-Vio 58.85% 0.31%

Anti-Oct3/4 A-APC Isoform -1 -1 -1 -1 -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ Anti-Sox2-FITC Anti-SSEA-5-VioBlue Anti-TRA-1-60-PE Anti-PSA-NCAM-APC

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 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 and double-positive (bold numbers) cells.

4 PSC cultures were also monitored visually by light Conclusion microscopy. The images show the typical morphology of PSC colonies (fig. 4A) or cells differentiated into neural The antibody panel described in this application note precursors (fig. 4B). 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 A B 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.

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