Flow cytometry provides a rapid and reliable method to quantify viable cells in a cell suspension. Determination of cell viability is critical when evaluating the physiological state of cells, such as in response to cytotoxic drugs and environmental factors, or during the progression of cancer and other disease states. In addition, it is often necessary to detect dead cells in a cell suspension in order to exclude them from analysis. Dead cells can generate artifacts as a result of nonspecific antibody binding or unwanted uptake of fluorescent probes. One method to identify the two cell populations is by dye exclusion. Live cells have intact membranes that exclude a variety of dyes that easily penetrate the damaged, permeable membranes of non-viable cells.
Several different fluorochromes can be used to stain non-viable cells including 7-amino actinomycin D (7-AAD). 7-AAD is a membrane impermeant dye that is generally excluded from viable cells. It binds to double stranded DNA by intercalating between base pairs in G-C-rich regions. 7-AAD can be excited at 488 nm with an argon laser. It has a relatively large Stokes shift, emitting at a maximum wavelength of 647 nm. Because of these spectral characteristics, 7-AAD can be used in combination with other fluorochromes excited at 488 nm such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE). The following protocol has been developed and optimized by R&D Systems Flow Cytometry Laboratory for the viability staining using 7-AAD.
Please read the protocol in its entirety before starting.
Note: Staining of surface antigens may be done at this point. 7-AAD cannot be used when labeling intracellular molecules.
Note: Use the FL-2 channel if staining only with 7-AAD. Collect 7-AAD fluorescence in the FL-3 channel if the cells have also been stained with an FITC- and/or PE-conjugated antibody.
Note: Do not wash cells after the addition of the 7-AAD staining solution.
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