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        FACS Analysis of ES Cells

        互联网

        1137

         

        • 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

         

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