Mario Roederer wrote: >This [acridine orange/ethidium bromide] is a fabulous way to do viability >testing! Once you do this >method, you will never do a trypan blue (yech) again. I learned to >do this in the Herzenberg laboratory at Stanford, brought it to the >VRC--and we've now incorporated it in our clinical trials. Every >time we thaw PBMC for doing immune function assays, we assess the >viability by fluorescence first (and, in fact, if viability is below >a treshhold, I think 60%, we discard the sample). We've even >developed an SOP for it. > >We use a combination of acridine orange and ethidium bromide (not >PI)--under a fluorescence scope, "green" is live and "red" is >dead--no ifs, ands, or buts--and easily scored by even the most green >students with risking a red face. > >In any case, our procedure is to prepare 3 mg/ml ethidium bromide in >absolute ethanol and 5 mg/ml acridine orange in ethanol. Store this >stock in a dark vial, refrigerated. To make a working solution, take >1 microliter of each added to 1 milliliter of PBS. This we store at >room temp by the fluorescence microscope, and make fresh every few >weeks. Please note that AO and EB are considered highly >carcinogenic: use gloves and a face mask when preparing the >concentrated stock solution, and use gloves when handling the working >solution. > >Dilute cells with an equal volume of the working solution and >immediately look on the fluorescence microscope (you can also dilute >1:10 if the cell count is too high). Remember to take this dilution >into account when you calculate original numbers. > >mr > >(PS, if you don't have EB, you could probably use PI at the same >concentration in its place.) One really should use PI, rather than EB, for dye exclusion tests of "viability"; EB can cross intact cytoplasmic membranes, but is normally pumped out of cells, while PI normally does not get into cells with intact membranes. EB works most of the time, but why take the chance? -Howard
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