DAPI and Intracellular Antigen Staining Protocol

 

  1. Harvest cells washing in PBS.
  2. Fix with 0.5% paraformaldehyde (PFA) for 15 min on ice.
  3. Wash twice with PBS.
  4. Fix pelleted cells in ice-cold 70% ethanol by adding with pasteur pipette on a vortex. Leave cells at 4oC for 30 mins.
  5. Pellet cells at approximately 2,000 rpm for 5 mins. Wash twice in PBS/0.25% saponin.
  6. Add 1 mg mcab and incubate for 30 min on ice.
  7. Wash twice in PBS/0.25% saponin.
  8. Add 0.5 mg mcab and incubate for 30 min on ice.
  9. Wash once in PBS/0.25% saponin.
  10. Add 1 mg/ml of DAPI (Sigma D9542).
  11. Analyse by flow cytometry collecting 25,000 events per sample.