Unless you are specifically studying apoptosis and cell death, you should include only healthy viable cells in your flow analysis and cell sorting experiments because dead and dying cells often show:
- Increased nonspecific antibody binding
- Increased autofluorescence
- Altered phenotype/epitopes
- Compromised RNA/DNA quality and proliferation capacity in downstream applications
Relying solely on forward and side scatter to identify healthy cells is not very accurate. Further, there are a wide variety of commercially available viability dyes to choose from so it is easy to fit them even into complex multicolor panels.
The specificity of common viability dyes is based on the integrity of cell membranes. Healthy cells exclude the dyes and stain only weakly if at all, whereas dying or dead cells have leaky membranes and the dye will penetrate into the cells giving rise to high intensity staining. It is important to titrate your viability dyes, just like your antibodies.
Viability dyes come in two flavors:
- DNA binding dyes (simple to use but cannot be combined with fixation or intracellular staining)
- Amine reactive dyes (may require extra steps in the staining protocol but can be combined with intracellular staining and fix/perm protocols)
Fig. 1. Cells were isolated from mouse spleen and stained for multiple lymphocyte markers as well as for viability. Live and dead (CD45+ CD3+) T cells could not be separated based on scatter alone.
Fig. 2. Artificially high CD4 counts due to nonspecific antibody binding to dead cells. Accurate flow analysis requires exclusion of dead cells.