In addition to Howard's comments. I use citric saline to detach my cells as it is less harsh than EDTA or trypsin and the cells reattach well.. I find I get excellent viability staining with this method. Recipe: For a 10x stock solution on citric saline, 1.35M potassium chloride 0.15M sodium citrate Autoclave and store at 4C, dilute 1:10 with sterile distilled water before use. Remove all media, flood flask bottom with 1x citric saline (prewarmed to 37C) incubate at 37C for maximum 5 minutes. Tap flask bottom to gently detach all cells. Decant cells, mix well for single cell suspension and add equal volumes PBS. Centrifuge and wash with fresh PBS. Cheers Lesley Mrs. Lesley Barber Edith Margaret Collie Centre for Blood Cell Therapies Div. Haematology/Medical Oncology Peter MacCallum Cancer Institute East Melbourne, 3002 VICTORIA , AUSTRALIA Tel 61 3 9656 1957 Fax 61 3 9656 1811 > -----Original Message----- > From: Howard Shapiro [SMTP:hms@shapirolab.com] > Sent: Wednesday, April 12, 2000 7:41 AM > To: Cytometry Mailing List > Subject: Re: Vital-staining > > > 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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