Viability of Ustilago maydis by Flow Cytometry

Amine-reactive dyes are useful for determining cell viability and are widely used in mammalian cells. These chromophores easily cross the damaged cell membranes, e.g., during apoptosis or autophagy processes, or due to physicochemical treatments, and thus can react irreversibly with the free amines in the cytosol. In live cells, chromophores react with exposed amines in the cell membrane but are excluded from the cytosol. Therefore, unfixed dye is washed after staining incubation, and the bound dye to dead cells remains associated. Since amine-reactive dyes are excited by lasers, dead cells can be clearly distinguished from live cells in a sample by flow cytometry. This protocol describes how to determine the percentage of dead and live cells in a population of Ustilago maydis by measuring the fluorescence emitted by the amine-reactive viability dye Ghost Dye™ Violet 450 using flow cytometry.