An Optimized LIVE/DEAD Assay Coupled with Flow Cytometry for Quantifying Post-Stress Survival in Yeast Cells

Summary

Here, we present a protocol to quantify post-stress survival in yeast samples. The assay employs two fluorescent dyes, SYTO 9 and propidium iodide (PI), to quantify plasma membrane integrity. It uses flow cytometry to provide quantitative and reproducible estimates of the live, dead, and damaged cell fractions for post-oxidative-stress samples.

Abstract

Quantifying survival is a common and critical task in yeast research. LIVE/DEAD stains based on plasma membrane integrity provide a rapid and high-throughput assay for yeast survival when coupled with flow cytometry. However, variations in staining buffer, dye concentration, incubation time, and flow cytometry settings can impact data quality and reproducibility. This protocol presents a standardized LIVE/DEAD assay for post-stress survival quantification in yeast using flow cytometry. After treating Candida glabrata, an opportunistic yeast pathogen, with different doses of hydrogen peroxide, the post-stress samples were stained with a two-component LIVE/DEAD stain consisting of SYTO 9 and propidium iodide (PI). Flow cytometry was used to distinguish live, damaged, and dead cell populations and quantify their percentages in each sample. Survival estimates based on the percent live statistic were compared to the Colony Forming Unit (CFU) result on the same sample. The two methods yielded consistent results for the mock- and lethal dose (1 M H₂O₂)-treated samples. At the sublethal dose of 100 mM H₂O₂, SYTO 9/PI estimated a higher survival rate than CFU, reflecting a key difference between the two, where the protocol presented here evaluates cell survival immediately after the stress, while CFU quantifies the percent of cells able to recover and reproduce. Hence, this protocol measures viability at an earlier stage of the cell-death process. In summary, the protocol described here provides a fast and scalable alternative to CFU for post-stress survival quantification in yeast. Its results provide complementary information to CFU by evaluating survival at an earlier stage and distinguishing between dead and damaged cells.

Introduction

The budding yeast subphylum contains many important biological and biotechnological models. A common and critical task in yeast research is quantifying the effect of genetic or environmental perturbations, the latter including stress or drug treatments. In industrial fermentation, for example, producers must monitor the viability of yeast cultures to ensure the efficiency and quality of the fermentation process^1,2. When studying pathogenic yeast species, measuring their survival after stress or antifungal treatment is critical for understanding the genetic and mechanistic basis for infection-related traits suc....

Protocol

The details of the reagents and the equipment used in this study are listed in the Table of Materials.

1. Application of hydrogen peroxide stress

1. At least a day before the experiment, innoculate an overnight culture of C. glabrata from a single colony on a freshly-streaked plate into 3 mL of Synthetic Complete (SC) media (1.7 g/L Yeast Nitrogen Base, complete amino acid mix, 2% w/v glucose, water), and culture it by placing the glass tube in a roller drum and grow at 30 °C. Alternatively, an orbital shaker can be used by placing the glass tube at a slanted angle and shaking the culture at 200 ....

Results

The SYTO 9/PI stain distinguishes live yeast cells from dead cells based on their fluorescence signals in the green (500 nm-560 nm) and red (>600 nm) channels. Live cells, which only accumulate SYTO 9, fluoresce strongly in the green channel, with minimal red signals. Dead cells accumulate both SYTO 9 and PI and are expected to fluoresce strongly in the red channel, with lower green fluorescence signals compared to live cells due to competitive exclusion of SYTO 9 by PI and FRET.

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Discussion

This protocol describes a LIVE/DEAD assay using two fluorescent dyes, SYTO 9 and PI, and using flow cytometry to quantify post-stress survival in yeast. While these two stains have been applied to assess survival in various yeast species in the literature and are available as a commercial kit, neither the manufacturer's manual nor the literature provides sufficient details on the staining protocol or flow cytometry settings⁷. Here, we present a standardized protocol resulting from systematic c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-Well Deep Well Plate	Thermo Fisher	278606	NUNC 96
Attune NxT flow cytometer with plate reader	Thermo Fisher A24858	A24858	Autosampler (A42901)
Candida glabrata			CG99, lab strain derived from clinical isolate (Cormack & Falkow, 1999)
FungaLight Yeast LIVE/DEAD Kit	Thermo Fisher A24858	L34952	Contains SYTO 9 (3.34 mM) and Propidium Iodide (20 mM). These two components can also be purchased separately from various vendors.
Hydrogen Peroxide	Sigma Aldrich	H1009
Leica Confocal SP8 Microscope	Leica	TCS SP8 X
Sodium Chloride	Sigma Aldrich	S9888	Used to make sterilized 0.85% NaCl solution
Yeast Nitrogen Base without amino acids	Sigma Aldrich	Y0626	used to make the Synthetic Complete media with 2% glucose and amino acid mix