Flow Cytometric Analysis Cell Cycle

Fixation

1) Collect 2 X 10⁶ cells.

2) Pellet cells by spinning at 1,000 rpm, 4°C for 5 minutes.

3) Resuspend cell pellet in 1 ml of cold PBS.

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

5) Store cells at -20°C 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 PBS.

7) Add 100 µl of 200 µg/ml DNase-free, RNaseA and incubate at 37°C for 30 minutes.

8) Add 100 µl of 1 mg/ml propidium iodide (light sensitive) and incubate at room temperature for 5-10 minutes.

9) Place samples in 12 X 75 Falcon tubes and read on Becton Dickinson FACStarPLUS.

Reference

Experimental Cell Research 207,142-151.