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Cell Cycle Analysis by PI Staining/FACS

Protocol 1.

Fixation

1) Collect 1 X 106 cells after proper treatment.

2) Pellet cells by spinning at 2,000 rpm, 4C for 5 minutes.

3) Resuspend cell pellet in 1 ml of cold FCM buffer (5mM EDTA in 1xPBS) .

4) Fix cells by adding 1 ml of -20C absolute ethanol.

5) Store cells at -20C in this fixation buffer until ready for analysis. (No more than 2 weeks)

Staining

6) Centrifuge (as above) fixed cells and resuspend pellet in 1 ml of FCM buffer.

7) Add 2 ul mg/ml DNase-free, RNaseA and incubate at 37C for 20 minutes.

8) Add 1 ul of 5 mg/ml propidium iodide (light sensitive) and incubate at room temperature for 15 minutes.

9) Pellet the cells by spinning at 2,000 rpm, 4C for 5 minutes.

10) Wash with FCM buffer once more, discard the buffer and resuspend the stained cells with 500ul FCM buffer.

11) Place samples in 12 X 75 Falcon tubes and read the data (% of G0 G1 G2 MS) on a FACS analysis system.

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