7-Aminoactinomycin D (7-AAD, catalog 163) is an intercalating red fluorescent reagent that binds between cytosine and guanine bases of DNA in membrane-compromised cells. This material, like its parent molecule, Actinomycin D, is a DNA-intercalator with growth-inhibitory properties. Normal healthy cells, with intact membrane structure, will exclude the polar 7-AAD vital dye. Mid to late apoptotic and necrotic cells will not exclude this dye and subsequently stain red when 7-AAD complexes with the nuclear DNA. As 7-AAD is membrane impermeant, it cannot reach the DNA in viable cells, thus allowing the identification of cells with permeabilized membranes in a population. 7-AAD distinguishes between living and dead cells by counterstaining nucleic acids red in necrotic, dead, dying, and membrane-compromised cells, while the DNA in healthy cells remains unstained.
7-AAD is supplied as a highly concentrated lyophilized powder at 0.26 mg/vial. Reconstitute it with 260 µL DMSO to yield a stock concentrate at 1 mg/mL. Add it to the cells at a final concentration of 5 µg/mL and analyze with a flow cytometer (Figure 1) or fluorescence microscope (Figure 2). When bound to nucleic acids, the maximum absorption is 546 nm and the maximum emission is 647 nm.
7-aminoactinomycin D may be used in combination with other fluorescent reagents for dual staining or multiplexing purposes. Use it with our FAM-FLICA® poly caspases inhibitor reagent (catalog 637) to include apoptotic cells in the analysis. When used together, these reagents will identify four populations of cells: living; early apoptotic; late apoptotic; and necrotic cells leading to more accurate results when assessing cell death (Figures 2 and 3).
- Reconstitute the vial of 7-AAD with 0.26 mL DMSO to create a stock concentrate at 1 mg/mL. Mix by swirling or tilting the vial, allowing the DMSO to travel around the base of the amber vial until completely dissolved. At room temperature, the reagent should be dissolved within a few minutes forming a red solution. 2. If storing the stock concentrate for future use, prepare small aliquots (50 µL) to avoid freeze-thaw cycles. The stock concentrate will be stable for 6 months when protected from light and stored at or below -20°C.
- Expose cells to the experimental conditions.
- Create 2 control tubes: untreated viable cells and untreated killed cells. These cells will be stained with 7-AAD to compensate the flow cytometer to ensure that dead cells shift along the FL3 axis. These controls will also determine the level of spontaneous cell death that normally occurs within the cell line when compared with the treated cells.
- Stain cells at a final concentration of 5 µg/mL of 7-AAD in the cell culture. This can be accomplished by pipetting the stock solution directly into the cell suspension at 1:200; e.g., add 2 µL stock to 400 µL cell suspension. This can also be accomplished by diluting the stock concentrate 1:10 to form the working solution, and then pipetting the working solution into the cells at 1:20. For example, add 50 µL 7-AAD stock concentrate into 450 µL PBS or sterile media. Mix by inverting or vortexing the vial at room temperature. Store on ice up to 2 hours. Then add the working solution to the cell suspension at approximately 1:20; e.g., put 25 µL diluted 7-AAD working solution into 475 µL cell suspension.
- Incubate 10-30 minutes on ice.
- If desired, wash cells twice with PBS and fix in 1% paraformaldehyde.
- Analyze with a flow cytometer: excitation at 546 nm; emission at 647 nm in FL3. Dead cells with compromised membranes will appear red.