This 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.) At 8:28 PM -0400 6/7/02, MSchwa3722@AOL.com wrote: >I was wondering if anyone uses these combinations of stains (acridine >orange/propidium iodide) to check viability of RBC containing samples using >flourescent microscopy. I had used this combination in the past but was >wondering if anyone had any suggestions on the relative concentrations of >each of the dyes and any references in the literature to this method of >checking viability. Your help as always, is greatly appreciated. > >Michael Schwartz, MT(ASCP) >Children's Hospital of Orange County >455 South Main St. >Orange, CA 92868 > >ph. - 714 532-8707
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