7-AAD is quite suitable for dead cell discrimination. It is excluded by viable cells and taken up by dead cells; apoptotic cells give intermediate staining (Schmid, I., Uitenbogaart, C.H. and Giorgi, J.V. (1994) Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry. Cytometry 15, 12-20). With 7-AAD you avoid problems with spectral overlap in the PE channel. To label the target cells, you could consider 5-chloromethylfluorescein diacetate (CMFDA), a tracker dye which is trapped within the cell. CMFDA diffuses freely into cells where it is converted by esterases into 5-chloromethylfluorescein. Reaction with protein thiols stabilizes the fluorescent dye in the cell. Another possibility is carboxyfluorescein acetate, succinimidyl ester (CFSE). Both of these compounds give fluorescein-type fluorescence. You will have to careful not too use too much label as they can be very bright and might tend to swamp the PE signal. I would have expected that the DiOC would be lost from the dead cells, in which case it would not allow you to identify dead targets. I do not know, but I would expect that CMFDA and CFSE should stay in the dead cells because they react with intracellular groups. Michael Ormerod 34 Wray Park Road Reigate RH2 ODE Telephone: voice & FAX: (0)1737 241726 Web site: http://ourworld.compuserve.com/homepages/Michael_Ormerod
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