Renate, PI is normally excluded from intact (living) cells, but I would imagine the EDTA is damaging the cell membranes enough to allow the PI to enter. This is also a big problem with cells that stick so well they have to be scraped off the flasks. I don't know that anyone has done this, but you might try EMA (ethidium monoazide), incubating the cells with it while they are still adherent. When irradiated by bright white light (fluorescent lamp) it irreversibly binds to the DNA, and the unbound dye will be flushed out of the living cells during a PBS wash step. Then you could try the EDTA removal. The original citation for EMA is Riedy et al, 1991. Cytometry 12:133-139, but after that I think you would be blazing new trails. I suppose you might want to use dishes instead of culture flasks (so you could remove the top during irradiation) but it might not matter. I hope this helps! Steve +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Steve G. Hilliard (706) 542-9474 University of Georgia Cell Analysis Facility flowman@uga.edu http://floweb.cb.uga.edu/ On Mon, 10 Apr 2000, Renate Wenig wrote: > > My name is Renate Wenig from the Paul-Ehrlich-Institut in Langen/Germany. > I'm FACS beginner. > Is there anybody with experience in vital-staining of adherent cells for > FACS-measerments? > 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? > Or can anybody say me where I can read about itMy E-mail adress is: wenre@pei.de > Thank you for answering! >
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