DNA Staining for Cell Cycle Analysis By flow Cytometry

Thursday, October 16, 2003

Description
This method of DNA staining utilizes ethanol to fix the cells and permeabilize the membrane, which allows the dye (Propidium Iodide) to enter the cells. Propidium Iodide (PI) is a DNA-binding fluorochrome that intercalates in the double-helix. Ribonuclease-A is used to eliminate the staining of double-stranded RNA.

Procedure
Fixation
Resuspend the cells in a 500 microliter volume of PBS and chill well on ice.

Prepare a 12x75 mm. tube containing 500 microliters of ice-cold 100% ethanol.

Rapidly pipet the cold cell suspension into the cold ethanol and mix by forcing air bubbles through the suspension. Pasteur-type transfer pipets work best for this.

Allow the suspension to remain on ice for 15 minutes.
The cells can remain in this state indefinitely if desired, but we find that clumping increases with the duration in alcohol. If it is necessary to pause the procedure here, make sure the tubes are securely sealed and kept refrigerated.

Debris Removal
Carefully underlayer the cell suspension with 1 ml ice-cold calf serum and centrifuge 3 minutes at 300 x g.

Carefully aspirate the liquid, taking care not to disturb the pellet. Using a cotton swab, carefully wipe the sides of the tube to remove any attached debris.

Add 2 ml. PBS. Vortex.

DNA Staining
Centrifuge 3 minutes at 300 x g. Remove as much liquid as possible but do not disturb the cell pellet, which is easily detached from the tube wall after ethanol fixation.

Add 125 microliters of RNase solution. Vortex.

Incubate in 37 degree C. water bath for 15 minutes.

Remove from water bath and add 125 microliters of PI stain solution. Vortex.

Allow to stand at room temperature for at least 30 minutes before analyzing on the flow cytometer.

The samples may remain at room temperature for up to 2 hours. Keep the cells on ice or refrigerate them if it is longer until your scheduled time on the flow cytometer.
Pipetting of the final cell suspension through a nylon monofilament mesh screen with 44 micron openings (we will supply these in our lab) immediately prior to analysis on the flow cytometer is highly recommended to remove large multicellular aggregates common in ethanol-fixed prepartions.

Recipes
DNA Stain: 5 milligrams of PI in 100 milliliters of 1.12% (w/v) of sodium citrate.

RNAse solution: 500 units per ml. in 1.12% (w/v) of sodium citrate.
Use a highly purified RNAse to avoid destruction fo the cells by contaminating proteases and/or DNAses. Otherwise, the RNase solution can be purified by heating to 75 degrees celcius for 30 minutes. Cool to room temperature before treating the cells.

100% ethanol.

Fetal or newborn bovine serum, (optional) for removal of debris.

Supplies

Tips
References
This method is based on, but modified from the following:

Braylan, RC, Benson, NA, Nourse, V, and Kruth, HS: Correlated analysis of cellular DNA, membrane antigens, and light scatter of human lymphoid cells. Cytometry, 2(5):337-343, 1982.

Crissman, HA, and Steinkamp, JA: Rapid simultaneous measurement of DNA, protein, and cell volume in single cells from large mammalian cell populations. J. Cell Biol., 59:766, 1973.

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DNA Staining for Cell Cycle Analysis By flow Cytometry