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.
- PBS (1X): 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4 or Hank’s Balanced Salt Solution (HBSS; 1X)
- Flow Cytometry Fixation Buffer (R&D Systems, Catalog # FC004, or an equivalent solution containing BSA and sodium azide)
- 7-AAD Staining Solution: 1 mg/mL 7-AAD in PBS (store at 2-8 °C in the dark)
- Detection Antibodies (optional)
- Isotype Control Antibodies (optional)
- FACS™ Tubes (5 mL round-bottom polystyrene tubes)
- Pipette Tips and Pipettes
- Harvest cells and aliquot up to 1 x 106 cells/100 μL into FACS tubes. Wash the cells 2 times by adding 2 mL PBS (or HBSS), centrifuging at 300 x g for 5 minutes, and then decanting the buffer from pelleted cells.
Note: Staining of surface antigens may be done at this point. 7-AAD cannot be used when labeling intracellular molecules.
- Resuspend cells in 100 μL of Flow Cytometry Staining Buffer.
- To adjust flow cytometer settings for 7-AAD, add 5 - 10 μL of 7-AAD staining solution to a control tube of unstained cells. Mix gently and incubate for 30 minutes at 4 °C in the dark.
- Determine 7-AAD fluorescence (using the FL-2 or FL-3 channel) with a FACScan™ instrument.
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.
- Acquire data for unstained cells and single-color positive controls.
- Add 5 - 10 μL of 7-AAD staining solution to each sample, and incubate for 30 minutes at 4 °C in the dark prior to analysis. Set the stop count on the viable cells from a dot-plot of forward scatter versus 7-AAD.
Note: Do not wash cells after the addition of the 7-AAD staining solution.
FACS and FACScan are trademarks of Becton Dickinson and Company.