Live cell imaging of mitochondrial redox state in mammalian cells and yeast

Methods Cell Biol. 2020:155:295-319. doi: 10.1016/bs.mcb.2019.11.008. Epub 2020 Feb 14.

Abstract

The redox state of mitochondria is determined by the levels of reducing and oxidizing species in the organelle, which reflects mitochondrial metabolic activity and overall fitness. Mitochondria are also the primary endogenous source of reactive oxygen species (ROS). This chapter describes methods to measure the mitochondrial superoxide levels and the redox state of the organelle in mammalian cells and yeast. We describe the use of dihydroethidium (DHE) and MitoSOX (a derivative of dihydroethidium bound to a lipophilic cation) to detect mitochondrial superoxide in yeast and mammalian cells, respectively. We also describe the use of genetically encoded fluorescent biosensors for quantitative analysis of mitochondrial NADPH levels (iNap) in mammalian cells and mitochondrial redox state (mito-roGFP) in yeast.

Keywords: Dihydroethidium (DHE); MitoSOX; NADPH; Oxidative stress; Reactive oxygen species (ROS); iNap3; roGFP.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biosensing Techniques
  • Cell Line
  • Cell Survival
  • Humans
  • Imaging, Three-Dimensional / methods*
  • Mammals / metabolism*
  • Mice
  • Mitochondria / metabolism*
  • Muscle Fibers, Skeletal / metabolism
  • NADP / metabolism
  • Oxidation-Reduction
  • Reactive Oxygen Species / metabolism
  • Saccharomyces cerevisiae / cytology*
  • Saccharomyces cerevisiae / metabolism
  • Superoxides / metabolism

Substances

  • Reactive Oxygen Species
  • Superoxides
  • NADP