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FACS Protocol

  • 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