Remove media and non-adherent cells from culture dish (and save if they are to be analyzed).
     Remove adherent cells from culture dish with trypsin being careful that the cells are harvested as soon as they begin
     detaching from the dish; do not leave them in trypsin any longer than necessary.
     Transfer trypsin-treated cells to conical centrifuge tubes.
     Centrifuge, 5 min, 1,000 rpm
     Pour off supernate and resuspend pellet in 1 ml PBS+PI buffer
     Perform cell count using a hemacytometer.
     Centrifuge cells again, 5 min, 1,000 rpm
     Pour off supernate and resuspend cells in PBS+PI (50 ug/ml) to a concentration of 1 x 106 cells/ml.
     Transfer 1 ml of cell suspension to a 15-ml conical centrifuge tube.
     Add 1 ml (equal volume) Vindelov's PI to each tube.
     Incubate 1 hr at 4C in the dark.
     Transfer to Falcon 2063 tubes for FACS analysis. May be kept at 4C O.N.
 
 

     REAGENTS
 
 

PBS + PI	filtered PBS + PI (50µg/ml)
Vindelov's Propidium Iodide Stain
1.21 g Tris base (0.01 M)	50.1 mg propidium iodide (7.5x10-5 M)
584 mg NaCl (10mM)	1.0 ml Igepal (equiv to Np-40)
10 mg Rnase (700 U)

     This recipe is per 1 liter. Adjust pH to 8.0 and filter through a 0.2µ filter. Wrap bottle in aluminum foil and store at 4C in the dark. Discard after one month.
 

     [Adapted from DNA Cell Cycle Analysis, Propodium iodide procedute, Handbook of Flow Cytometry Methods, J. Paul
     Robinson, Ed., Wiley Liss, 1993. This article further references, Krishan, A., Rapid flow cytometric analysis of
     mammalian cell cycle by propidium iodide staining, J. Cell Biol., 66:188-193, 1975]