Measurement of Oxidative Stress in Cells Exposed to Hypoxia and Other Changes in Oxygen Concentration

This chapter discusses different methods for measuring oxidative stress in yeast cells exposed to anoxia. Reliable assays involve measuring oxidative DNA and protein damage, and measuring the expression of the oxidative stress-induced gene SOD1. These assays should be useful for other types of cells exposed to hypoxia or anoxia. It is also found that carboxy-H2-dichlorodihydrofluorescein diacetate,—a commonly used fluorescein derivative— is useful for assessing relative ROS levels in yeast cells grown at different steady-state oxygen concentrations and in comparing levels of oxidative stress in respiration-proficient and respiration-deficient cells, but it is not useful for assessing ROS levels when cells are shifted from one oxygen concentration to another and followed for long periods of time. Protocol for exposing normoxic yeast cultures to hypoxic or anoxic conditions , detection of 8-OH-dG in mitochondrial and nuclear DNA and induction of SOD1 is also discussed.