7AAD

7-Aminoactinomycin D, commonly abbreviated as 7-AAD, is a fluorescent dye used to stain double-stranded DNA in flow cytometry. It is a cell-impermeant molecule that can only enter cells with compromised plasma membranes, making it useful as a viability indicator. In staining protocols, live cells exclude 7-AAD, whereas dead or late-apoptotic cells take up the dye and exhibit bright red fluorescence. The dye binds to DNA and yields fluorescence in the red/orange region of the spectrum, with an emission maximum near 647 nm, typically excited by a 546 nm (green-yellow) laser or, on some instruments, by a 488 nm laser.

7-AAD is widely employed in conjunction with other fluorochromes in multicolor flow cytometry and is also applicable to fixed or permeabilized cells for assessment of DNA content and cell cycle distribution. Because it stains DNA rather than specific cell types, it is useful for distinguishing intact from non-viable cells in heterogeneous samples and for combining viability assessment with surface-marker analyses in a single panel. The staining protocol usually uses short incubation times at room temperature and protection from light to prevent photobleaching. RNase treatment is typically unnecessary for 7-AAD because it primarily binds double-stranded DNA, though some protocols may include RNase to minimize RNA fluorescence in certain contexts.

Limitations include potential overlap with other red-emitting fluorochromes in multicolor panels and the need for proper compensation. 7-AAD remains one of several standard viability dyes used in flow cytometry to quantify cell viability and DNA content.