FACSAnalysisofESCells

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