PROPIDIUM IODIDE STAINING FOR CELL CYCLE (DNA) ANALYSIS BY FACS

PROCEDURE A

Krishan introduced ploidy analysis using propidium iodide.  Later his technique was modified by the addition of NP-40.  Procedures for both the original and the modification are described below.

Krishan Procedure [*]

1.    Resuspend 1 x 106  cells in 1 ml of P.I. solution (0.05 mg/ml propodium iodide in 0.1% sodium citrate)

2.    Incubate 5 to 10 min at 4 degrees C

3.    Analyze by flow cytometry

Modified Krishan Precedure [**]

1.    Resuspend 1 x 106 cells in 1 ml of modified Krishan staining buffer; vortex 10 secs.

2.    Incubate at least 30 minutes on ice; centrifuge

3.    Resuspend pellet in 1 ml fresh modified Krishan staining buffer

4.    Syringe through 27g needle

5.    Filter through 37 micron nylon mesh immediately before flow analysis.

        Modified Krishan buffer
                0.1% sodium citrate
                0.3% NP-40
                0.05 mg/ml propidium iodide
                0.02 mg/ml RNase
 

Ref.
    [*]  A. Krishan, Rapid cytoflurorometric analysis of mammalian cell cycle by propidium iodide staining, J. Cell. Biol.  66:188-193.

    [**]  L.G. Dressler, et. al., 1988, DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens, Cancer 61:420-427.