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.

2

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.

Cell surface staining Intracellular staining

Sample BufferAnti-TRA-

1-60-PECD15-PE-

Vio770, humanAnti-SSEA-4-

VioGreenAnti-SSEA-5-

VioBlueInside Perm

Anti-Sox2-FITC

Anti-Oct3/4 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.

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

lati

ve c

ell n

umb

er

10³-1 10¹ 10²0 1

REA Control (S)-PE

Re

lati

ve c

ell n

umb

er

10³-1 10¹ 10²0 1

REA Control (I)-APC

Re

lati

ve c

ell n

umb

er

10³-1 10¹ 10²0 1

REA Control (S)-VioGreen

Re

lati

ve c

ell n

umb

er

10³-1 10¹ 10²0 1

Mouse IgG1-VioBlue

Re

lati

ve c

ell n

umb

er

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

4

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.

A B

Miltenyi Biotec B.V. & Co. KG | Friedrich-Ebert-Straße 68 | 51429 Bergisch Gladbach | Germany | Phone +49 2204 8306-0 | Fax +49 2204 85197 macs@miltenyibiotec.de | www.miltenyibiotec.com

Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact.

V.03

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. autoMACS, MACS, MACSQuant, REAfinity, StemMACS, Vio, VioBlue, VioGreen, and the MACS logo are registered trademarks or trademarks of Miltenyi Biotec and/or its affiliates in various countries worldwide. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2020 Miltenyi Biotec and/or its affiliates. All rights reserved.