Dead cells may compromise flow cytometric data analysis by non-specifically binding antibodies; therefore it is important to exclude dead cells from the analysis. This is done by adding a DNA binding dye. Either propidium iodide (PI), 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI), 7-amino-actinomycin D (7-AAD), DRAQ7, SYTOX ADDVanced, or amine-reactive dyes can be used (article).
The flow cytometric analysis of cell viability may be challenging when infected and human cells (BSL2 samples) or intracellular antigens i.e. cytokines and transcription factors) are analyzed, since these types of samples require fixation before the flow acquisition. Use of the new amine-reactive dyes (Invitrogen, BD Horizon™) allows the discrimination of dead cells by pre-incubation of cells with dye before fixation (see article). Additionally, the new antraquinone dye CyTRAK Orange can also label live or fixed/dead cells.
Other commercially available kits may exist.
- Resuspend cells in 0.5 ml 1X PBS. If doing extracellular staining, after the last wash (DO NOT USE FIXED CELLS) resuspend cells in 0.5 ml 1X PBS.
- Add (final concentration) 1 µg/ml PI, 500-1000 ng/ml DAPI, 2.5 µm 7-AAD, or 5.0 µM CyTRAK Orange.
- Shortly after addition of the viability marker, collect events on cytometer:
- PI: use 488 nm laser, collect using 610/20 BP
- DAPI: optimal 355 nm laser, collect using 440/40 BP; may use 405 nm laser, collect using 450/50 BP
- 7-AAD: use 488 nm laser, collect using 670/14 BP (may work 610/20 BP)
- CyTRAK Orange: use 488 nm laser, collect using 610/20 BP