Amine‐Reactive Dyes for Dead Cell Discrimination in Fixed Samples

Abstract

Amine-reactive dyes, also known as LIVE/DEAD fixable dead cell stains, are a class of viability dyes suitable for identifying dead cells in samples that will be fixed. These dyes cross the cell membranes of dead cells, and react with free amines in the cytoplasm. Live cells exclude these dyes because their cell membranes are intact, and free dye is washed away after staining. Notably, the reaction is irreversible; therefore, when cells are fixed and permeabilized (as with intracellular staining procedures), the bound dye remains associated with the dead cells (unlike other viability dyes). Since amine-reactive dyes are fluorescent when excited by lasers, dead cells can be identified by flow cytometry. This unit describes procedures, troubleshooting, and outcomes for using the two most commonly used amine-reactive dyes, ViViD and Aqua Blue.