Zielonka 2010 Free Radic Biol Med

Zielonka J, KalyanaramanB (2010) Hydroethidine- and Mito-SOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: Another inconvenient truth. Free Radic Biol Med 48:983–1001.

» PMC3587154 Open Access

Zielonka J, KalyanaramanB (2010) Free Radic Biol Med

Abstract: Hydroethidine (or dihydroethidium) (HE) is the most popular fluorogenic probe used for detecting intracellular superoxide radical anion. The reaction between superoxide and HE generates a highly specific red fluorescent product, 2-hydroxyethidium (2-OH-E+). In biological systems, another red fluorescent product, ethidium (E+), is also formed, usually at a much higher concentration than 2-OH-E+. In this article, we have reviewed the methods to selectively detect the superoxide-specific product (2-OH-E+) and the factors affecting its levels in cellular and biological systems. The most important conclusion of the present review is that it is nearly impossible to assess the intracellular levels of the superoxide specific product, 2-OH-E+, using the confocal microscopy or other fluorescence-based microscopic assays and that it is essential to measure by HPLC the intracellular HE and other oxidation products of HE, in addition to 2-OH-E+, in order to fully understand the origin of red fluorescence. The chemical reactivity of mitochondria-targeted hydroethidine (Mito-HE, MitoSOX Red ®) with superoxide is similar to the reactivity of HE with superoxide and therefore, all of the limitations attributed to the HE assay are applicable to Mito-HE (or Mito-SOX) as well.

• Bioblast editor: Iglesias-Gonzalez J

Cited by

• Komlódi T, Gnaiger E (2022) Discrepancy on oxygen dependence of mitochondrial ROS production - review. MitoFit Preprints 2022 (in prep).

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Stress:Oxidative stress;RONS 

MitoFit 2022 ROS review