The University of Iowa Carver College of Medicine

Flow Cytometry Facility

DNA Staining with PI

The following protocol is for DNA staining with propidium iodide for cell cycle analysis (complex hypotonic solution).

Reagents

  • Stock Solution
  • 20mg/ml propidium iodide (in 1X PBS) [store refrigerated, protected from light]
  • 10X Lysis Buffer
  • 10mM TRIS (1.211g/L in dd H2O) [MW=121.1g]
  • 1mM EDTA (0.3722g/L in dd H2O) [EDTA Disodium Sulfate2H2O MW= 372.2g]
  • 1% Triton X-100
  • Store at room temperature
  • Hypotonic Staining Solution (for 50ml)
  • 50mg sodium citrate
  • 5ml lysis buffer
  • 45ml ddH2O
  • Mix until sodium citrate dissolved
  • 125µl stock propidium iodide solution
  • Store refrigerated and protected from light [good for at least 2 weeks]

Staining

  1. Pellet 2 x 106 cells.
  2. Carefully aspirate supernatant leaving as little buffer as possible without aspirating cells.
  3. Add 1ml hypotonic propidium iodide staining solution.
  4. Vortex.
  5. Store on ice for at least 15 minutes before running on flow cytometer.