LIVE/DEAD Yeast Viability Kit Protocol

This kit combines a novel two-color fluorescent probe for yeast viability, FUN 1, with a fluorescent fungal surface labeling reagent, Calcofluor White M2R. Plasma membrane integrity and metabolic function of fungi are required to convert the yellow-green fluorescent intracellular staining of FUN 1 into red-orange intravacuolar structures; Calcofluor white M2R labels cell-wall chitin with blue-fluorescence, regardless of metabolic state.

1. Preparation of yeast suspensions

1. Grow yeast to late log phase (107–108 cells/mL) in medium (e.g., YPD)

2. Add 50 µL of the yeast culture to 1 mL of wash buffer

3. Centrifuge at 10,000 × g for 5 minutes

4. Remove supernatant and resuspend pellet in 1 mL of wash buffer

2. Staining yeast

1. Add 1 µL of Component A (FUN 1 cell stain) and 5 µL of Component B (Calcofluor white M2R) to 1 mL of yeast suspension (10 µM and 25 µM final concentrations of each of the dyes, respectively)

2. Incubate at 30°C in the dark for 30 minutes

3. Put 5 µL of the stained yeast suspension onto a glass slide and cover with a coverslip

4. Image cells with the appropriate filters listed below

Saccharomyces cerevisiae stained with FUN 1 cell stain, which generates red-fluorescent intravacuolar structures, and with Calcofluor white M2R, a blue-fluorescent cell wall stain.