Flow cytometry

  1. Discard media.
  2. Add PBS 6mL to wash.
  3. Discard PBS.
  4. Treat trypsin 2mL and incubate at 37C for 3min.
  5. Add media 10mL.
  6. Transfer to 15mL conical tube.
  7. Centrifuge 1000g for 5min at 4C.
  8. Discard supernatant.
  9. Add PBS 6mL to wash.
  10. Centrifuge 1000g for 5min at 4C.
  11. Discard PBS.
  12. Add 5% cold FBS in PBS 1mL.
  13. Count cells and harvest 2 x 10^6 cells.
  14. Transfer to amber 1.5mL EP tube.
  15. Centrifuge 1000g for 5min at 4C.
  16. Discard 5% FBS in PBS.
  17. Turn light off and add cold 5% FBS in PBS 100uL.
  18. Pipette to resuspend cells.
  19. Incubate 10min on ice.