FIXATIONandDNAStainingforCellCycleAnalysis

Background

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.

Generally, the method can be combined with the procedures for direct labeling or indirect labeling of cell surface antigens. Use only FITC-labeled antibodies since PI emits in the orange to red region of the spectrum (FITC emits green). And do not fix the cells with paraformaldehyde - start with this protocol after the last wash step in the direct or indirect immunofluorescence protocols.

The fluorescence from many antibody-labeled cell surface antigens is often partially diminished following this procedure. Other antigens may be totally denatured and all fluorescence may be lost. It is best to test for this before beginning a large dual-staining (cell surface and DNA) experiment.

Materials

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

   
   

2. 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.

3. 100% ethanol.

   
   

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

   
   

5. Cells in suspension, counted and viability-checked. The procedure is written for aliquots of 1 million cells, but you can scale it up as required. The cells should be suspended in culture media or PBS without serum, as the ethanol fixation step will percipitate the proteins in the serum.

   This procedure does not necessarily require 100% viable cells, but generally the more dead cells you have, the more debris and multi-cell aggregates you can expect.

   
   

Equipment

1. Centrifuge. You should know how the RPM translates into G-force.

   
   

2. Precision adjustable micropipet. It should cover the range from 100 to 1000 microliters.

   
   

3. Pasteur-type transfer pipettes, either glass or plastic.

   
   

4. Cotton swabs.

   
   

5. Vortex mixer.

   
   

6. 37 degree C. water bath.

   
   

7. 12x75 mm polystyrene tubes. The clear plastic kind. If you can, buy the Falcon brand because they fit the instrument best. If you can't, don't worry - we will supply them when you bring your samples to the lab.

   
   

8. Ice bucket with cover. Generally, cells are more stable and tolerate insult better when they're cold. The ice bucket cover keeps light out, which could bleach the fluorescent dye.

   
   

9. Flow cytometer. If you analyze your samples in our lab, the instrument you use will most likely be a FACScan or FACSort, made by Becton-Dickinson. (See our instrument list for more details.)

   
   

Procedure

Fixation

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

   
   

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

   
   

3. 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.

   
   

4. 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

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

   
   

6. 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.

   
   

7. Add 2 ml. PBS. Vortex.

   
   

   DNA Staining

8. 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.

   
   

9. Add 125 microliters of RNase solution. Vortex.

   
   

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

    
    

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

    
    

12. 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.