7-AAD

7-AAD is a cell-impermeant, G-C affinity DNA stain with an excitation peak at 549 nm and an emission peak at 648 nm.

7-aminoactinomycin (7-AAD) is an intercalating dye that binds to double-stranded DNA (dsDNA). It has a strong preference for G-C regions, in contrast to other DNA stains, such as DAPI (A-T affinity) and propidium iodide (no sequence specificity). Because 7-AAD is cell impermeable, it cannot be used for live cell staining, unlike Hoechst 33342 or acridine orange, which are membrane permeable. As such, 7-AAD is most often used to label cells with compromised plasma membranes, such as fixed, necrotic or otherwise nonviable cells, serving as a viability dye for flow cytometry applications.

The most common application of 7-AAD is flow cytometry. In particular, it can be used for cell cycle determination, cell sorting and studying of apoptosis. For apoptosis studies, it is frequently paired with an annexin v conjugate (e.g. Annexin V FITC), wherein annexin v identifies early apoptosis through binding to translocated phosphatidylserine while 7-AAD marks necrotic cells with damaged membranes.

7-AAD vs PI

7-AAD often serves as a replacement for propidium iodide (PI). Both fluorophores are membrane impermeant, staining non-viable or fixed cells. Likewise, both can be excited by the 488 nm argon laser line. One difference is that propidium iodide has a much larger quantum yield (0.2) versus 7-AAD (0.02). This results in a brighter fluorescence signal, on average, for PI. On the other hand, 7-AAD has a significantly large Stokes’ shift. This reduces spectral overlap in multiplex analysis, making 7-AAD more compatible with common fluorophores such as FITC and phycoerythrin (PE).

7-AAD has a molecular weight of 1270.43 daltons. It has an excitation wavelength of 549 nm and an emission wavelength of 648 nm. It is well excited by the 488 nm argon laser line. It can be read using a flow cytometer with the PE/Cy5 or FL3 channel.

A 1 to 10 mM stock solution of 7-AAD can be prepared using DMSO. The stock solution is good for 6 months, if stored in a dark location at < -20 °C.