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PMID: 20116425 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Review

Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth.

Free radical biology & medicine ·Vol. 48 ·No. 8 ·2010-04-15 ·Pages 983-1001

Zielonka J, Kalyanaraman B

Abstract

Hydroethidine (HE; or dihydroethidium) 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, is also formed, usually at a much higher concentration than 2-OH-E(+). In this article, we review 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 this review is that it is nearly impossible to assess the intracellular levels of the superoxide-specific product, 2-OH-E(+), using 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(+), 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 MitoSOX) as well.

MeSH Terms
Chromatography, High Pressure Liquid Culture Media Cytochromes c/chemistry Drug Stability Electrophoresis Ethidium/analogs & derivatives,analysis False Positive Reactions Fluorescent Dyes/metabolism Hydrogen Peroxide/chemistry Metalloporphyrins/chemistry Microscopy, Confocal/methods Mitochondria/metabolism Phenanthridines/chemistry Reactive Oxygen Species/analysis Superoxides/analysis,metabolism Ultrasonics
Chemicals
2-hydroxyethidium 5-(6'-triphenylphosphoniumhexyl)-5,6-dihydro-6-phenyl-3,8-phenanthridinediammine Culture Media Fluorescent Dyes Metalloporphyrins Phenanthridines Reactive Oxygen Species Superoxides hydroethidine Cytochromes c Hydrogen Peroxide Ethidium
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Zielonka Jacek
Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. balarama@mcw.edu
Kalyanaraman B
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Article Info
Journal
Free radical biology & medicine
Abbr.
Free Radic Biol Med
ISSN
1873-4596
DOI
10.1016/j.freeradbiomed.2010.01.028
Email
balarama@mcw.edu
Published
2010-04-15
Epub
2010-00-29
Pages
983-1001
Language
English
Region
United States
NLM ID
8709159
PMCID
PMC3587154
Subset
IM
Grants
NHLBI NIH HHS · R01 HL073056 · United States
NHLBI NIH HHS · R01 HL073056-05 · United States
NHLBI NIH HHS · HL063119 · United States
NHLBI NIH HHS · R01 HL063119 · United States
NHLBI NIH HHS · R01 HL063119-10 · United States
NINDS NIH HHS · R01 NS040494-08 · United States
NINDS NIH HHS · R01 NS040494 · United States
NINDS NIH HHS · NS40494 · United States
NHLBI NIH HHS · HL073056 · United States
External Links
PubMed source DOI: 10.1016/j.freeradbiomed.2010.01.028 PMC Full Text View journal details
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