Multicolor flow cytometry analysis of human pluripotent stem cell cultures

Multicolor flow cytometry analysis of human pluripotent stem cell cultures Background Antibody Thorough characterization of pluripotency is mandator...
Author: Clara Merritt
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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

Multicolor flow cytometry analysis of hPSCs  |  February 2016

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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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