Immunophenotyping of the pluripotency status of human pluripotent stem cells (PSCs) using flow cytometry
Miltenyi Biotec-tested panel 25 (MBTP 25)
Miltenyi Biotec-tested panel 25 (MBTP 25)
This application protocol describes the simultaneous quantification of intracellular and surface markers for easy monitoring of the pluripotency status of human PSC cultures using flow cytometry.
Protocol
Surface staining
- Volumes given below were for up to 106 nucleated cells. When working with fewer than 106 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×106 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
- Cell number was determined.
- Cells were centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- A master mix with all antibodies was prepared according to the dilutions mentioned in the respective antibody data sheets.
- 100 µl antibody master mix was added to the cells.
- Cells were resuspended and incubated for 10 minutes in the dark at 4 °C.
- Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes at 4 °C. Supernatant was aspirated completely.
- Intracellular staining was performed.
Intracellular staining
- Reagents were prepared freshly. Failure to do so may lead to sub-optimal results.
- The required total buffer volumes were calculated beforehand; volumes will depend on the number of cells to be analyzed as well as the number of tests to be performed.
- Cells previously stained for surface markers were resuspended in 250 µL of Inside Fix solution from the Inside Stain Kit.
- Cells were mixed well and incubated for 20 minutes at room temperature.
- Cells were centrifuged for 5 minutes at 300×g.
- Supernatant was aspirated completely and the pellet was resuspended in 1 mL of PEB buffer.
- Cells were centrifuged for 5 minutes at 300×g and supernatant was aspirated completely.
- Intracellular antibodies were diluted in Inside Perm according to the respective data sheet to a final volume of 100 µL and added to the cells.
- Cell suspension was incubated for 10 minutes at room temperature.
- 1 mL of Inside Perm buffer was added and cells were centrifuged for 5 minutes at 300xg.
- Supernatant was aspirated completely and the pellet was resuspended in 0.5-1 mL of PEB buffer.
- Flow cytometric analysis was performed using the MACSQuant® Analyzer.
View details
Materials
Antibody panel details
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| TRA-1-60 | REA157 | Pluripotency | PE | 585/40 (blue) |
| CD15 | VIMC6 | Differentiation | PE-Vio® 770 | 750LP (blue) |
| SSEA-4 | REA101 | Pluripotency | VioGreen™ | 525/50 (violet) |
| SSEA-5 | 8e11 | Pluripotency | VioBlue® | 450/50 (violet) |
| Oct3/4 Isoform A | REA338 | Pluripotency | APC | 655-730 (red) |
| Sox2 | REA320 | Pluripotency | FITC | 525/50 (blue) |
Additional reagents used
- Phosphate-buffered saline (PBS), pH 7.2
- PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA.
- Deionized or distilled water
- Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343) or MACSQuant X (# 130-105-100)
- Inside Stain Kit (# 130-090-477)
- (Optional) MACSQuant Calibration Beads (# 130-093-607)
- (Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)
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REA338
ICFC
USD 140.00
REA320
ICFC, MICS, IF, IHC
USD 94.00
REA101
FC
USD 131.00
