Propidium Iodide Staining for FACS

Propidium Iodide Staining for Fluorescence-Activated Cell Sorting (FACS)

  1. Collect 2x106 cells.
  2. Resuspend cell pellet in 2 ml PBS.
  3. Vortex gently while adding 5 ml of 95% ethanol.
  4. Fix at least 30 minutes at RT.
  5. Store at 4°C (or -20°C) until ready to stain.


  1. Spin cells out of fix (3000x 5 minutes).
  2. Resuspend pellet in 1 ml of PI Working Solution.
  3. Add 100 µl of 1 mg/ml DNase-free Rnase.
  4. Incubate at 37°C for 30 minutes.
  5. Samples are ready for flow cytometry (FCM) analysis. They may be stored in refrigerator O/N.


Note: For a single FCM run, use the same PI working solution to reduce variation between samples.

Stock Propidium Iodide (PI)

  • 500 µg/ml made in H20

Working Solution

  • 1:10 dilution with PBS or Tris buffer