Cell cycle analysis using propidium iodide

Reagents

• Propidium Iodide Staining Solution: 3.8 mM sodium citrate, 40 ug/ml PI [Sigma, P 4170] in PBS.
• RNase A stock solution: 10 ug/ml RNase A [Worthington Biochemicals, RASE LS005649, LS005650] (boiled for 5 min, aliquoted and stored frozen at -20 degrees C).
• 100% ethanol stored in the freezer at -20 °C.
• Wash buffer: PBS + 0.1% Bovine albumin or serum.

Protocol

1. Harvest cells and prepare single cell suspension in wash buffer
2. Wash cells twice and resuspend at 1-2 x 10⁶ cells/ml (see note 3)
3. Aliquot 1 ml cells in a 15 ml polypropylene, V-bottomed tube on ice and allow to cool. Add 3 ml cold (-20 °C) absolute ethanol. The ethanol can be added forcibly by expelling from a pipette or dropwise while vortexing. Determine the best method for each cell type to minimize clumping and cell loss (see note 2).
4. Fix cells for at least 1 hour at 4 °C. Fixation overnight is preferable. (Cells may be stored in the ethanol fixative at -20 °C for several weeks prior to PI staining and flow cytometric analysis).
5. Wash cells twice with PBS. It may be necessary to centrifuge cells at a slightly higher rcf to pellet after ethanol fixation as the cells become flocculent.
6. Add 1 ml of propidium iodide staining solution to cell pellet and mix well. Add 50 µl of RNase A stock solution and incubate 3 hr at 4 °C (see note 1)
7. Stained samples may be stored for up to a week at 4 °C, protected from light.
8. Do NOT wash prior to analysis.

Notes

1. You can speed up the PI/RNAse step by incubating at room temp (~40 mins) or at 37 °C for ~20 mins. The incubation step is to ensure that the RNase has digested all the RNA, which otherwise would interfere with the DNA signal. PI at this concentration stains very rapidly.
2. Adding the cold ethanol dropwise is probably the best way to go for fixation. The idea is that you don't ramp up the ethanol concentration too fast (this can lead to aggregation problems). As you get more ethanol in the tube, you can add it ever faster. Methanol may be used instead of ethanol.
3. It's important to have similar concentrations of cells in each tube. If there are large variations, the intensity of staining will be similarly variable.
4. FACScan settings: FL2 = 400V. Turn on DDM, set to FL2. FL2-A gain approx 3. FL2-W gain approx 1.25. Gate on FL2-width versus FL2-area to exclude aggregates. Analyze FL2-A histograms with FlowJo.
5. IMPORTANT: If your cells contain GFP, this protocol will remove all the GFP fluorescence unless the GFP is anchored inside the cell (to membranes, or histones for instance). If you can't anchor your GFP, try a brief fixation with 0.5% paraformaldehyde before the first step.

References

Crissman HA, 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.

Krishan A. Rapid flow cytofluorometric analysis of cell cycle by propidium iodide staining. J. Cell Biol., 66:188, 1975.

http://southmed.usouthal.edu/com/biotechweb/rcl.procedurea.html http://southmed.usouthal.edu/com/biotechweb/rcl.procedureb.html http://southmed.usouthal.edu/com/biotechweb/rcl.procedurec.html http://southmed.usouthal.edu/com/biotechweb/rcl.techpoints.html http://www.meduniwien.ac.at/user/johannes.schmid/PIstaining3.htm http://www.ueb.cas.cz/Olomouc1/LabDol/Research/Flow_cytometry/Protocols/Two-step_proc.htm http://www.med.umich.edu/flowcytometry/PDF%20files/Pi.pdf http://www.med.umich.edu/flowcytometry/PDF%20files/HYPOpi.pdf