Methods to monitor ROS production by fluorescence microscopy and fluorometry

Methods Enzymol. 2014;542:243-62. doi: 10.1016/B978-0-12-416618-9.00013-3.


Mitochondria are considered one of the main sources of reactive oxygen species (ROS). The overgeneration of ROS can evoke an intracellular state of oxidative stress, leading to permanent cell damage. Thus, the intracellular accumulation of ROS may not only disrupt the functions of specific tissues and organs but also lead to the premature death of the entire organism. Less severe increases in ROS levels may lead to the nonlethal oxidation of fundamental cellular components, such as proteins, phospholipids, and DNA, hence exerting a mutagenic effect that promotes oncogenesis and tumor progression. Here, we describe the use of chemical probes for the rapid detection of ROS in intact and permeabilized adherent cells by fluorescence microscopy and fluorometry. Moreover, after discussing the limitations described in the literature for the fluorescent probes presented herein, we recommend methods to assess the production of specific ROS in various fields of investigation, including the study of oncometabolism.

Keywords: Cytosol; Hydrogen peroxide; Mitochondria; Reactive oxygen species; Respiratory chain; Superoxide anion.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Ethidium / analogs & derivatives
  • Ethidium / metabolism
  • Fluorenes / metabolism
  • Fluorescent Dyes / metabolism
  • Fluorometry / methods*
  • Humans
  • Microscopy, Fluorescence / methods*
  • Mitochondria / metabolism
  • Organophosphorus Compounds / metabolism
  • Oxazines
  • Permeability
  • Phenanthridines / metabolism
  • Proteins / analysis
  • Reactive Oxygen Species / analysis*
  • Reactive Oxygen Species / metabolism


  • Fluorenes
  • Fluorescent Dyes
  • MitoSox Red
  • Organophosphorus Compounds
  • Oxazines
  • Phenanthridines
  • Proteins
  • Reactive Oxygen Species
  • dihydroethidium
  • Amplex Red
  • Ethidium