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Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future. J Biol Chem 2018 Jun 29;293(26):10363-10380



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Scopus ID

2-s2.0-85049623107 (requires institutional sign-in at Scopus site)   76 Citations


Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O2̇̄), hydrogen peroxide, lipid hydroperoxides, peroxynitrite, and hypochlorous and hypobromous acids play a key role in many pathophysiological processes. Recent studies have focused on mitochondrial ROS as redox signaling species responsible for promoting cell division, modulating and regulating kinases and phosphatases, and activating transcription factors. Many ROS also stimulate cell death and senescence. The extent to which these processes occur is attributed to ROS levels (low or high) in cells. However, the exact nature of ROS remains unknown. Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria. However, because most of these redox-active probes (untargeted and mitochondria-targeted) are oxidized by several ROS species, attributing redox probe oxidation to specific ROS species is difficult. It is conceivable that redox-active probes are oxidized in common one-electron oxidation pathways, resulting in a radical intermediate that either reacts with another oxidant (including oxygen to produce O2̇̄) and forms a stable fluorescent product or reacts with O2̇̄ to form a fluorescent marker product. Here, we propose the use of multiple probes and complementary techniques (HPLC, LC-MS, redox blotting, and EPR) and the measurement of intracellular probe uptake and specific marker products to identify specific ROS generated in cells. The low-temperature EPR technique developed to investigate cellular/mitochondrial oxidants can easily be extended to animal and human tissues.

Author List

Cheng G, Zielonka M, Dranka B, Kumar SN, Myers CR, Bennett B, Garces AM, Dias Duarte Machado LG, Thiebaut D, Ouari O, Hardy M, Zielonka J, Kalyanaraman B


Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University
Gang Cheng PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin
Micael Joel Hardy PhD Visiting Assistant Professor in the Biophysics department at Medical College of Wisconsin
Balaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of Wisconsin
Suresh Kumar PhD Associate Professor in the Pathology department at Medical College of Wisconsin
Jacek M. Zielonka PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Aconitate Hydratase
Cell Line
Electron Transport Complex I
Electron Transport Complex III
Energy Metabolism
Enzyme Inhibitors
Molecular Probe Techniques
Oxidative Stress
Reactive Oxygen Species