7-AAD Red Fluorescent Live/Dead Stain

1 Citation

7-Aminoactinomycin D (7-AAD) is a red fluorescent vital dye that intercalates in double-stranded DNA with a high affinity for GC-rich regions. 7-AAD does not pass through intact cell membranes. It may also be used to label all cells after fixation.

Figure 1. Jurkat cells were untreated (live) (A) or treated with 90% ethanol (dead) (B). Cells were pelleted by centrifugation (200 x g for 5 minutes) and resuspended in PBS. Cells were then stained with 7-AAD for 10 minutes on ice, and analyzed using an Accuri C6 flow cytometer in FL-3. Only 4% of untreated cells (A) are dead compared with 97.6% of the treated cells (B) (ICT 226:30-31).
Figure 2. Jurkat cells were exposed to staurosporine to induce apoptosis, then dually stained with the green fluorescent FAM-FLICA poly caspase probe to detect apoptosis via caspase activity, and the red dye 7-AAD to detect necrosis. Early apoptotic cells fluoresce green with FAM-FLICA. Dually stained green and red fluorescing cells are in mid to late stage apoptosis (active caspases and compromised cell membranes). Necrotic cells fluoresce red. (ICT 196:70).
Figure 3. Jurkat cells were treated with DMSO (A) or staurosporine (B), then stained with poly caspase FAM-FLICA apoptosis reagent (Cat. 637) and 7-AAD. FAM-FLICA was analyzed on FL-1, and 7-AAD on FL-3. Four cell populations are identified - see key for details. FAM-FLICA detects cells in early apoptosis (lower right quadrant), which 7-AAD cannot detect alone. Only 10.8% of control cells were apoptotic (LR: 4.5% +UR: 6.3%) compared with 97.1% of induced cells (B; LR: 46.4% + UR: 50.7%) (ICT).
Figure 1. Jurkat cells were untreated (live) (A) or treated with 90% ethanol (dead) (B). Cells were pelleted by centrifugation (200 x g for 5 minutes) and resuspended in PBS. Cells were then stained with 7-AAD for 10 minutes on ice, and analyzed using an Accuri C6 flow cytometer in FL-3. Only 4% of untreated cells (A) are dead compared with 97.6% of the treated cells (B) (ICT 226:30-31).
Figure 1. Jurkat cells were untreated (live) (A) or treated with 90% ethanol (dead) (B). Cells were pelleted by centrifugation (200 x g for 5 minutes) and resuspended in PBS. Cells were then stained with 7-AAD for 10 minutes on ice, and analyzed using an Accuri C6 flow cytometer in FL-3. Only 4% of untreated cells (A) are dead compared with 97.6% of the treated cells (B) (ICT 226:30-31).
Figure 2. Jurkat cells were exposed to staurosporine to induce apoptosis, then dually stained with the green fluorescent FAM-FLICA poly caspase probe to detect apoptosis via caspase activity, and the red dye 7-AAD to detect necrosis. Early apoptotic cells fluoresce green with FAM-FLICA. Dually stained green and red fluorescing cells are in mid to late stage apoptosis (active caspases and compromised cell membranes). Necrotic cells fluoresce red. (ICT 196:70).
Figure 3. Jurkat cells were treated with DMSO (A) or staurosporine (B), then stained with poly caspase FAM-FLICA apoptosis reagent (Cat. 637) and 7-AAD. FAM-FLICA was analyzed on FL-1, and 7-AAD on FL-3. Four cell populations are identified - see key for details. FAM-FLICA detects cells in early apoptosis (lower right quadrant), which 7-AAD cannot detect alone. Only 10.8% of control cells were apoptotic (LR: 4.5% +UR: 6.3%) compared with 97.1% of induced cells (B; LR: 46.4% + UR: 50.7%) (ICT).

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Data Sheet Safety Data Sheet

SKU: 6163

Size: 0.26 mg
Price:
$172.00

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).

546 nm / 647 nm
Flow cytometry, Fluorescence microscope
2-8°C
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United States
  1. 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.
  2. Expose cells to the experimental conditions.
  3. 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.
  4. 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.
  5. Incubate 10-30 minutes on ice.
  6. If desired, wash cells twice with PBS and fix in 1% paraformaldehyde.
  7. Analyze with a flow cytometer: excitation at 546 nm; emission at 647 nm in FL3. Dead cells with compromised membranes will appear red.

Product Specific References

PMID Publication
38443460Goswami, N, et al. 2024. EVATOM: an optical, label-free, machine learning assisted embryo health assessment tool. Communications biology, 268.

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