Fluorescence Detection of Increased Reactive Oxygen Species Levels in Saccharomyces cerevisiae at the Diauxic Shift.

ABSTRACT

The budding yeast Saccharomyces cerevisiae is a facultative organism that is able to utilize both anaerobic and aerobic metabolism, depending on the composition of carbon source in the growth medium. When glucose is abundant, yeast catabolizes it to ethanol and other by-products by anaerobic fermentation through the glycolysis pathway. Following glucose exhaustion, cells switch to oxygenic respiration (a.k.a. "diauxic shift"), which allows catabolizing ethanol and the other carbon compounds via the TCA cycle and oxidative phosphorylation in the mitochondria. The diauxic shift is accompanied by elevated reactive oxygen species (ROS) levels and is characterized by activation of ROS defense mechanisms. Traditional measurement of the diauxic shift is done through measuring optical density of cultures grown in a batch at intermediate time points and generating a typical growth curve or by estimating the reduction of glucose and accumulation of ethanol in growth media over time. In this manuscript, we describe a method for determining changes in ROS levels upon yeast growth, using carboxy-H(2)-dichloro-dihydrofluorescein diacetate (carboxy-H(2)-DCFDA). H2-DCFDA is a widely used fluorescent dye for measuring intracellular ROS levels. H2-DCFDA enables a direct measurement of ROS in yeast cells at intermediate time points. The outcome of H2-DCFDA fluorescent readout measurements correlates with the growth curve information, hence providing a clear understanding of the diauxic shift.