PI Labeling Method for FACS Analysis

  1. Trypsinize cells in a 5 mL petri-plate with 1 mL of trypsin.
  2. Add 5 mL PBS and spin cells at 800 rpm for 5 mins.
  3. Add 5 mL of 70% EtOH (cold) while vortexing gently. Make sure that cells are dispersed in the ethanol and not clumpy. If clumpy, gently disperse cells using a pipetman.
  4. Leave cells in EtOH at 4°C O/N or 4 hours minimum for fixing.
  5. Next day spin cells at 800 rpm for 5 mins and resuspend cells in 5 mL PBS, wait 1 min and spin again at 800 rpm for 5 mins.
  6. Resuspend cells in 50 µL PBS, plus 3.3 µL RNAse (30 mg/mL) and incubate at 4°C for 5 minutes.
  7. Add 450 µL FACS buffer (PBS + 2% FBS) and 25 µL PI (1 mg/mL). Incubate at 4°C for 30 minutes.
  8. Analyze immediately after incubation at 4°C, in order to prevent clumping of the cells.