FACS Analysis of ES Cells
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1048
- 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
