- Isolate cells and dissociate to single cell suspension (can use Gibco Cell Dissociation Buffer, Accutase or Trypsin)
- Wash with 10% FBS/DMEM:F12
- For surface markers, resuspend in appropriate volume of 10% FBS/DMEM:F12 and keep on ice.
- For intracellular markers, resuspend in 1ml 4% paraformaldehyde/PBS and incubate for 10min at room temperature
- Wash with 10% FBS/DMEM:F12
- Resuspend in 0.1% Triton X-100/PBS and incubate for 15min on ice
- Wash with 10% FBS/DMEM:F12
- Resuspend in an appropriate volume of 10% FBS/DMEM:F12 and keep on ice
- Add 50μl cell suspension to 50μl 10% FBS/DMEM:F12 containing appropriate amount of antibody to give the final concentration listed below and incubate for 30min to 1 hour
- Wash twice with 10% FBS/DMEM:F12
- Resuspend in 100μl of 10% FBS/DMEM:F12 containing secondary antibody (AlexaFluor 488 conjugate) at 1:100
- Incubate for 30min
- Wash twice with 10% FBS/DMEM:F12
- Resuspend in appropriate volume of 10% FBS/DMEM:F12
| Antibody | Dilution | Source | Catalog Number |
|---|---|---|---|
| SSEA1 | 1:100 | Santa Cruz | sc-21702 |
| SSEA4 | 1:100 | Santa Cruz | sc-21704 |
| Tra-1-60 | 1:100 | Kind gift of Peter Andrews, Sheffield University | |
| Tra-1-81 | 1:100 | Kind gift of Peter Andrews, Sheffield University | |
| Oct-4 | 1:20 | Santa Cruz | sc-5279 |