Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2014 Jan 10;20(2):372-82.
doi: 10.1089/ars.2012.4886. Epub 2012 Oct 19.

Methods for detection of mitochondrial and cellular reactive oxygen species

Sergey I Dikalov  1 , David G Harrison
Affiliations
Review

Methods for detection of mitochondrial and cellular reactive oxygen species

Sergey I Dikalov et al. Antioxid Redox Signal. .

Abstract

Significance: Mitochondrial and cellular reactive oxygen species (ROS) play important roles in both physiological and pathological processes. Different ROS, such as superoxide (O2(•-)), hydrogen peroxide, and peroxynitrite (ONOO(-)), stimulate distinct cell-signaling pathways and lead to diverse outcomes depending on their amount and subcellular localization. A variety of methods have been developed for ROS detection; however, many of these methods are not specific, do not allow subcellular localization, and can produce artifacts. In this review, we will critically analyze ROS detection and present advantages and the shortcomings of several available methods.

Recent advances: In the past decade, a number of new fluorescent probes, electron-spin resonance approaches, and immunoassays have been developed. These new state-of-the-art methods provide improved selectivity and subcellular resolution for ROS detection.

Critical issues: Although new methods for HPLC superoxide detection, application of fluorescent boronate-containing probes, use of cell-targeted hydroxylamine spin probes, and immunospin trapping have been available for several years, there has been lack of translation of these into biomedical research, limiting their widespread use.

Future directions: Additional studies to translate these new technologies from the test tube to physiological applications are needed and could lead to a wider application of these approaches to study mitochondrial and cellular ROS.

PubMed Disclaimer

Figures

<b>FIG. 1.</b>
FIG. 1.
Dichlorodihydrofluorescein diacetate (DCFH-DA) intracellular reactions and redox cycling of 2,7-dichlorodihydrofluorescein (DCF).
<b>FIG. 2.</b>
FIG. 2.
Detection of hydrogen peroxide (H2O2) with N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red).
<b>FIG. 3.</b>
FIG. 3.
Detection of O2•−-specific products of dihydroethidium (DHE) and mitoSOX. (A) Formation of DHE O2•−-specific product 2-hydroxyethidium, and (B) mitochondrion-targeted accumulation of mitoSOX and detection of mitochondrial O2•−.
<b>FIG. 4.</b>
FIG. 4.
Selective activation of cytoplasmic NADPH oxidases with PMA or treatment of mitochondria with antimycin A results in site-specific production of O2•−. Site-specific detection of cytoplasmic and mitochondrial O2•− using DHE (A) and mitoSOX (B). *p<0.05 vs control.
<b>FIG. 5.</b>
FIG. 5.
Chemical structures of hydroxylamine probes (A), ESR detection of cellular superoxide (B), hydrogen peroxide (C) and detection of mitochondrial superoxide (D).
<b>FIG. 6.</b>
FIG. 6.
Molecular structures and reactivity of boronate probes.
See this image and copyright information in PMC

References

    1. Abou-Khalil S, Abou-Khalil WH, Planas L, Tapiero H, and Lampidis TJ. Interaction of rhodamine 123 with mitochondria isolated from drug-sensitive and -resistant Friend leukemia cells. Biochem Biophys Res Commun 127: 1039–1144, 1985 - PubMed
    1. Ahmad R, Som S, Johnson DH, Zweier JL, Kuppusamy P, and Potter LC. Multisite EPR oximetry from multiple quadrature harmonics. J Magn Reson 214: 135–143, 2012 - PMC - PubMed
    1. Albrecht SC, Barata AG, Grosshans J, Teleman AA, and Dick TP. In vivo mapping of hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity of redox homeostasis. Cell Metab 14: 819–829, 2011 - PubMed
    1. Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, and Lukyanov S. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3: 281–286, 2006 - PubMed
    1. Benov L, Sztejnberg L, and Fridovich I. Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical. Free Radic Biol Med 25: 826–831, 1998 - PubMed

Publication types