Unit

UNIT 25.1 High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells

  1. Puja Billis,
  2. Yvonne Will,
  3. Sashi Nadanaciva

Published Online: 19 FEB 2014

DOI: 10.1002/0471140856.tx2501s59

Current Protocols in Toxicology

Current Protocols in Toxicology

How to Cite

Billis, P., Will, Y. and Nadanaciva, S. 2014. High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells. Current Protocols in Toxicology. 59:25.1:25.1.1–25.1.14.

Author Information

  1. Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc, Groton, Connecticut

Publication History

  1. Published Online: 19 FEB 2014

Abstract

Reactive oxygen species (ROS) are constantly produced in cells as a result of aerobic metabolism. When there is an excessive production of ROS and the cell's antioxidant defenses are overwhelmed, oxidative stress occurs. The superoxide anion is a type of ROS that is produced primarily in mitochondria but is also generated in other regions of the cell including peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. Here, a high-content imaging assay using the dye dihydroethidium is described for identifying compounds that generate superoxide in eukaryotic cells. A high-content imaging assay using the fluorescent dye tetramethylrhodamine methyl ester is also described to identify compounds that impair mitochondrial membrane potential in eukaryotic cells. The purpose of performing both assays is to identify compounds that (1) generate superoxide at lower concentrations than they impair mitochondrial membrane potential, (2) impair mitochondrial membrane potential at lower concentrations than they generate superoxide, (3) generate superoxide and impair mitochondrial function at similar concentrations, and (4) do not generate superoxide or impair mitochondrial membrane potential during the duration of the assays. Curr. Protoc. Toxicol. 59:25.1.1-25.1.14. © 2014 by John Wiley & Sons, Inc.

Keywords:

  • oxidative stress;
  • superoxide;
  • dihydroethidium;
  • mitochondrial membrane potential;
  • TMRM;
  • high-content imaging