Good question! We do not see a lot of green when using FDA and our 'scope with freshly isolated mouse spleen cells. If we raise the FDA conc. to 20 ug/ml in the sample, leave it in >5 min, and shut off all the lights, we can see faint green. However, cultured (cloned) T cells glow bright green when we harvest them from flasks--and they are resting-state T cells, capable of being stimulated with specific Ag (I should add they are CD4 TH1-type helper T cells from mouse kept in culture with periodic Ag restimulation). Freshly-isolated unstimulated human WBC glow fairly well (Histopaque isolation) for us. We only use FDA for counting on the 'scope. Best, Beverly Beverly E. Barton, Ph.D. Assistant Professor Department of Surgery/Division of Urology UMDNJ-NJMS MSB G519 185 S. Orange Avenue Newark, New Jersey 07103 Telephone 973-972-0662 E-mail bartonbe@umdnj.edu Telefacsimile 973-972-3892 On Feb 15, 2005, at 1:46 PM, Uriel TK wrote: > Dear friends: > I wanted to know if anyone has used fluorescein diacetate in resting T > cells, specifically murine thymocytes, as a "viability" probe. We want > to do 3ple stains without changing our system and PI and 7AAD are not > compatible with our current panel. Protocols state the need for active > esterase activity and i don't know if resting t cells are "active > enough" for FDA to work well. > > Many thanks for the help, > > Uriel.Received on Fri Feb 18 12:58:00 2005
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