Renate Wenig writes- >I want to count absolute cell-number of vital cells with FACS. >Until now I try to stain the dead cells with PI but it doesn't work: >After detaching cells from the flaks bottom with EDTA all cells are PI >positiv in FACS. >Can anybody give me useful tips or instructions for vital-staining of >adherent cells? Many of the PI-positive cells you see after detachment are probably dead; it is possible that all of them are. Can you plate them after you detach them, which would establish viability? The problem here is that procedures used to detach cells from flasks may permeabilize the membrane, transiently or permanently. If the permeabilization is permanent, the cells are dead; if transient, they can recover. If you don't need to keep the cells viable (as for sorting) after the flow cytometric analysis, but only want to know the fraction or number of cells which were viable while they were in the flask, you should use a staining procedure before detaching the cells. There are several which would work; you might try 10-25 ug/mL 7-aminoactinomycin D (7-AAD)(excitation 488 nm, emission 675 nm). This will only get into the dead cells. Then, wash the cells and detach them, with 10-25 ug/ml plain actinomycin D added to all wash solutions and to the final suspending medium. Actinomycin D is nonfluorescent, and will bind to DNA in the cells which are permeabilized by the detachment and other preparative procedures; the cells which were originally dead will contain much more 7-AAD than those which were not, so you will know what the viable fraction was before you started abusing the cells. This trick is based on work by Terry Fetterhoff et al and Ingrid Schmid et al, among others; there are other techniques you could use, but this is what I'd try first. -Howard
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