Propidium iodide DNA staining protocol

Prepare in advance

1. RNASE solution: 2 mg/mL RNAse A (Bovine Pancreas Type II, Sigma) in HBSS. Divide into 500 ul aliquots and freeze.
2. Propidium iodide: 0.1 mg/mL PI (Sigma) in HBSS with 0.6% NP-40.
3. 1 x PBS with 2% FBS.

Procedure

1. Prepare cell suspensions.
2. Adjust concentration to 2 x 10⁶ cells/mL, transfer to conical tubes and centrifuge at 1100 rpm (300 g) for five minutes.
3. Resuspend cells in 500uL PBS. Fix cells by adding 1 mL ice cold 70% ethanol dropwise. Vortex gently while adding ethanol. Fix on ice for at least 2 hours.
4. Centrifuge for five minutes and aspirate the supernatant. Wash once with PBS (may have 2% FBS).
5. Resuspend pellet in 500 uL 0.1 mg/mL propidium iodide (Sigma) with 0.6% NP-40. Gently vortex to mix.
6. Add 500 uL of 2 mg/ml RNAse solution to cells. Gently vortex and incubate cells in dark at room temperature for 30 minutes.
7. Filter and keep cold and in dark until analysis. Cells are analyzed in this DNA staining solution.

Reference: Nicoletti et al. J Imm Meth 1991;139.