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Surface Antigens and DNA: Hypotonic Propidium Iodide Staining

This protocol is ideal for surface labeled cells.

Stock solutions (stored at 4°C)

1 mg/ml trisodium citrate
1 mg/ml RNAse in Phosphate Buffered Saline (PBS)

Note: You cannot boil PBS!

10% Triton X-100 (v/v in H2O)
500 mg/ml propidium iodide (PI Solution, Roche Cat. No. 1 348 639)

Working solution

(for 100 samples; prepare weekly, store at 4°C)

5.0 ml RNAse
500 ml 10% Triton X-100
2.0 ml PI Solution
42.5 ml trisodium citrate

Staining procedure

(after last antibody incubation)

  1. Wash 106 cells with protein-free PBS, aspirate supernatant and break up pellet.
  2. Incubate 10 min on ice.
  3. Add 500 ml ice cold Working Solution and mix gently.
  4. Incubate overnight on ice (in refrigerator).


Brons PPT, Van Erp PEJ, Pennings AHM. Flow Cytometry: Methods in Cell Biology, Volume 41, Chapter 6, page 97. (1994)