Flow cytometry

D-2 Cell culture (25T or 75T / 60mm dish)

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