FACS for determination of cell cycle distribution (PI, i.e. propidium iodide, staining)

1.  Harvesting of the cells. Some people use trypsin, but incubation for 5 min. at 37 ˚C in PBS with 0.1 % EDTA may be preferable since it is more gentle to the cells. It is important to have intact cells which are not clumped together. It is essential if you have cell populations that easily come off the dish (e.g. apoptotic or mitotic) to collect the cell medium and pool this together with the cells coming off the dish. To collect the cells from the dish it is most convenient to use a pasteur pipet equipped with a yellow bulb. Be sure you disperse the cells by gently squeezing the cells up and down in the pasteur pipet.

2.  Centrifuge in a tabletop centrifuge at 1200 rpm for 5 min. (4 ˚C)

3.  Aspirate supernatant carefully and wash once in 5 ml PBS 1 % FCS.

4.  Permeablization of cells. Aspirate the supernatant once again and resuspend cell pellet thoroughly in 200 µl PBS. Add dropwise while vortexing at low speed 800 µl of 95 % (or 100 %) ethanol. It’s important to add the ethanol slowly to prevent clumping.

5.  The cells can be stored at this point at 4 ˚C even for weeks until final PI staining and analysis.

6.  Centrifuge in a tabletop centrifuge at 1200 rpm for 5 min. (4 ˚C) and aspirate ethanol solution. Wash once with 5 ml PBS 1 % FCS, centrifuge again and carefully aspirate without disturbing the cell pellet.

7.  PI staining: Make up the following PI staining solution: 50 µg/ml PI, 10 mM Tris pH 7.5, 5 mM MgCl2, 10 µg/ml RNase (guaranteed DNase free). For 10 ml, mix the following: 1 ml 0.5 mg/ml PI stock soln., 100 µl 1 M Tris pH 7.5, 50 µl 1 M MgCl2, 20 µl RNase A, 8.9 ml dH2O

8.  Add 1 ml of PI staining solution for each sample (if using cells from a p100 dish; when using a p60 dish, 0.5 ml is sufficient) and mix by pipetting up and down. Incubate at 37 ˚C for 30 min. The cells are now ready for analysis and should be analyzed within 24 hrs. Until analysis, keep the cells on ice with the tubes wrapped in aluminium foil, since PI is light sensitive. You have to transfer your cells in the end to Falcon 2054 tubes !

PI stock soln. is 10x: 0.5 mg/ml PI in 0.038M citrate pH 7.0 (stored at 4 ˚C)