Dear Robert, It depends what you want to do with the 7-AAD! 7-AAD is a G-C specific intercalating dye which, although it has a high DNA binding constant, does not bind irreversibly with DNA. That is why when you wash it you may reduce background but you will loose staining intensity as the 7-AAD leaks out of the cell. If you are interested in directly staining cells and using 7-AAD only as a marker of live/dead discrimination then you do not need to wash the cells. We use it routinely at 1ug/ml final concentration for CD34 analysis in a no wash technique which does not give significant background staining. For a reference see Keeney et al Cytometry, Comm. in Clin Cytometry volume 34,#2 April 15, 1998. If you have a requirement to permeabilise your cells after staining with 7-AAD an excellent article recently published by Ingrid Schmid in Cytometry 35:64-74, 1999 will provide the information you need. This paper looked at cell surface phenotype and DNA cell cycle position of viable and non viable cells in a T cell leukemia cell line. They added non fluorescent Actinomyicin D to prevent the fluorescent 7-AAD from diffusing from the positive dead cells to permeabilised negative live cells. It's a good article. Mike Michael Keeney Technical Specialist Hematology/Flow Cytometry London Health Sciences Centre 800 Commissioners Road East London, Ontario Canada N5X 2Z2
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