Cited by 125Redox biology and oxidative stressNitric Oxide and Endothelin EffectsAntioxidant Activity and Oxidative Stress

Measurement of reactive oxygen species (ros) and mitochondrial ros in ampk knockout mice blood vessels

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Wang, Qilong, Zou, Ming-Hui·2018·Methods in Molecular Biology

Abstract

Reactive oxygen species (ROS) are a group of unstable and highly reactive molecules or free radicals typically generated as by-products of cellular processes involving molecular oxygen. In vascular cells, the excessive ROS generation results in the initiation and progression of cardiovascular diseases (CVD). Therefore, a dynamic, robust, and accurate ROS detection method in the blood vessels is essential for pathophysiological research studies of the cardiovascular system.In this chapter, we describe a fluorescence dye-based detection method for assaying superoxide and mitochondrial superoxide in mouse aorta using dihydroethidium (DHE) and MitoSOX. The protocol includes preparation of frozen aortic tissue sections, monitoring DHE oxidation-derived fluorescence by fluorescence microscopy, and high-performance liquid chromatograph-based analysis of MitoSOX and its oxidation products. For studying the role of AMP-activated protein kinase (AMPK) in the redox regulation, we employed AMPKα2 knockout mice and observed increased superoxide and mitochondrial superoxide levels in the aorta of AMPK knockout mice relative to the wild-type group. This novel ROS detection method will be valuable for investigating the roles of cellular and/or mitochondrial ROS in the pathogenesis of CVDs.

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  • DHE reacts rather specifically with cellular O2●−, producing ethidium and 2-hydroxyethidium; it crosses membranes and can be oxidized anywhere, so only total cellular O2●− production can be detected; DNA binding can cause false localization interpretations.

    “Dihydroethidium (DHE) is a red fluorescent probe that rather specifically reacts with cellular O2●−, resulting in the two fluorescent products ethidium and 2-hydroxyethidium. Only 2-hydroxyethidium is generated after reaction with O2●−, while ethidium is formed by nonspecific redox reactions. However, DHE easily crosses cellular membranes and therefore can be oxidized by O2●− anywhere in and”

    From Functions of ROS in Macrophages and Antimicrobial Immunity (2021) · cites this paper as [360]

  • MitoSOX accumulates in the mitochondrial matrix and specifically detects O2●−, but can be oxidized by other ROS while diffusing into mitochondria; Nox2 can cause false positive signals.

    “MitoSOX Red (MitoSOX) is a modified DHE analog attached to a triphenylphosphonium (TPP) group, which leads to accumulation of the probe in the mitochondrial matrix [365,366]. Like DHE itself, MitoSOX specifically detects O2●− and emits red fluorescence after the reaction (see Figure 7) [30,99,360]. In contrast to DHE, MitoSOX does so mainly in the mitochondrial matrix, though. Notably, MitoSOX can”

    From Functions of ROS in Macrophages and Antimicrobial Immunity (2021) · cites this paper as [360]

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Authors
Wang, Q., & Zou, M.-H.
Affiliations
Georgia State University (US)
Corresponding author
Zou, Ming-Hui
Venue
Methods in Molecular Biology
Published
2018
Volume
1732
Pages
507-517
ISSN
  • 1064-3745 (ISSN-L)
  • 1940-6029
OpenAlex
W2789049383
Cited by
125
References
18
Citations by year
2026: 10 · 2025: 13 · 2024: 15 · 2023: 19 · 2022: 24 · 2021: 19 · 2020: 18 · 2019: 4 · 2018: 2
FWCI
14.44
Type
book chapter
Language
en
Open access
Locations
  • Methods in molecular biology (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institute on Aging (R01AG047776)
  • National Heart, Lung, and Blood Institute (R01HL105157, R01HL079584, R01HL142287, R01HL137371, R01HL132500, R01HL074399, R01HL080499, R01HL140954, R01HL110488, R01HL128014, R01HL089920, R01HL096032)
  • National Cancer Institute (R01CA213022)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Health Sciences › Medicine › Biochemistry › Antioxidant Activity and Oxidative Stress
Keywords
reactive oxygen species, mitochondrial superoxide, superoxide, AMPK, aorta, dihydroethidium, MitoSOX, cardiovascular disease, redox regulation, vascular cells
MeSH terms
Animals, Aorta, Cardiovascular Diseases, Chromatography, High Pressure Liquid, Ethidium, Fluorescent Dyes, Microscopy, Fluorescence, Mitochondria, Oxidation-Reduction, Phenanthridines, Reactive Oxygen Species, Mice, Knockout, Myocytes, Smooth Muscle, Endothelial Cells, Mice, AMP-Activated Protein Kinases

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