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

Quantifying survival is a common and critical task in yeast research. LIVE/DEAD stains based on plasma membrane integrity provide a rapid and ...

University of Iowa

Stanford University

Voltage-gated sodium (Na) channels play a central role in the generation and propagation of action potentials in excitable cells such as neurons and muscles. To determine how the phenotypes of Na-channel mutants are affected by other genes, we performed a forward genetic screen for dominant modifiers of the seizure-prone, gain-of-function  Na-channel mutant,  Our analyses using chromosome deficiencies, gene-specific RNA interference, and single-gene mutants revealed that a null allele of  () dominantly suppresses  phenotypes. Reduced  function also suppressed phenotypes of other seizure-prone Na-channel mutants,  and  Notably,  mutants expressed 50% less  than wild-type flies, further supporting the notion that  and  interact functionally. Introduction of a loss-of-function  mutation into a  background led to up- and down-regulation of various genes, with those encoding cytochrome P450 (CYP) enzymes most significantly over-represented in this group. Because  is a fly ortholog of mammalian hematopoietic prostaglandin D synthase, and in mammals CYPs are involved in the oxygenation of polyunsaturated fatty acids including prostaglandins, our results raise the intriguing possibility that bioactive lipids play a role in -mediated suppression of  phenotypes.

Reduced Function of the Glutathione S-Transferase S1 Suppresses Behavioral Hyperexcitability in  Expressing Mutant Voltage-Gated Sodium Channels.

The goal of this investigation was to examine how individual variation in inattention and hyperactivity is related to motor timing difficulties and whether children's performance on simple laboratory timing tasks is related to their performance on a virtual road-crossing task using a head-mounted virtual reality display system.

Do Inattention/Hyperactivity and Motor Timing Predict Children's Virtual Road-Crossing Performance?

Acquired stress resistance (ASR) enables organisms to prepare for environmental changes that occur after an initial stressor. However, the genetic basis for ASR and how the underlying network evolved remain poorly understood. In this study, we discovered that a short phosphate starvation induces oxidative stress response (OSR) genes in the pathogenic yeast  and protects it against a severe HO stress; the same treatment, however, provides little benefit in the low pathogenic-potential relative, . This ASR involves the same transcription factors (TFs) as the OSR, but with different combinatorial logics. We show that Target-of-Rapamycin Complex 1 (TORC1) is differentially inhibited by phosphate starvation in the two species and contributes to the ASR via its proximal effector, Sch9. Therefore, evolution of the phosphate starvation-induced ASR involves the rewiring of TORC1's response to phosphate limitation and the repurposing of TF-target gene networks for the OSR using new regulatory logics.

Divergence of TORC1-mediated Stress Response Leads to Novel Acquired Stress Resistance in a Pathogenic Yeast.

Acquired stress resistance (ASR) enables organisms to prepare for environmental changes that occur after an initial stressor. However, the genetic basis for ASR and how the underlying network evolved remain poorly understood. In this study, we discovered that a short phosphate starvation induces oxidative stress response (OSR) genes in the pathogenic yeast C. glabrata and protects it against a severe H2O2 stress; the same treatment, however, provides little benefit in the low pathogenic-potential relative, S. cerevisiae. This ASR involves the same transcription factors (TFs) as the OSR, but with different combinatorial logics. We show that Target-of-Rapamycin Complex 1 (TORC1) is differentially inhibited by phosphate starvation in the two species and contributes to the ASR via its proximal effector, Sch9. Therefore, evolution of the phosphate starvation-induced ASR involves the rewiring of TORC1's response to phosphate limitation and the repurposing of TF-target gene networks for the OSR using new regulatory logics.

Divergence of TORC1-mediated stress response leads to novel acquired stress resistance in a pathogenic yeast.

Quantifying post-stress survival in yeasts is crucial for biological, biomedical, and industrial research. Traditional methods like Colony Forming Unit (CFU) assays are labor-intensive and time-consuming. In this study, we systematically characterize a two-dye (SYTO 9 / Propidium Iodide) LIVE/DEAD assay coupled with flow cytometry to rapidly and scalably quantify post-stress survival in diverse yeast species. By optimizing staining buffer, dye concentrations and staining time, we minimized artifacts and improved reproducibility. Notably, we identify an "Intermediate" population containing damaged cells with enhanced SYTO 9 uptake but little propidium iodide (PI) accumulation under sublethal stress, providing finer gradations of cellular damage compared to CFU or PI staining alone. We demonstrate the assay's applicability across , , and  after hydrogen peroxide treatment and in  after Amphotericin B exposure. While CFU is more sensitive at lower stress levels, the SYTO 9/PI staining effectively distinguishes sublethal from lethal doses, offering a valuable alternative for rapid, high-throughput survival quantification.

Article	Year
Reduced Function of the Glutathione S-Transferase S1 Suppresses Behavioral Hyperexcitability in Expressing Mutant Voltage-Gated Sodium Channels. G3 (Bethesda, Md.)\| PubMed ID: 32054635	2020
Do Inattention/Hyperactivity and Motor Timing Predict Children's Virtual Road-Crossing Performance? Journal of pediatric psychology\| PubMed ID: 34402519	2021
Divergence of TORC1-mediated Stress Response Leads to Novel Acquired Stress Resistance in a Pathogenic Yeast. bioRxiv : the preprint server for biology\| PubMed ID: 37781605	2023
Divergence of TORC1-mediated stress response leads to novel acquired stress resistance in a pathogenic yeast. PLoS pathogens\| PubMed ID: 37871123	2023
An Optimized LIVE/DEAD Assay Using Flow Cytometry to Quantify Post-Stress and Antifungal-Treatment Survival in Diverse Yeasts. bioRxiv : the preprint server for biology\| PubMed ID: 40502067	2025

Hanxi Tang

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