Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo

Michael P Murphy¹, Hülya Bayir^{2

3}, Vsevolod Belousov⁴, Christopher J Chang⁵, Kelvin J A Davies⁶, Michael J Davies⁷, Tobias P Dick⁸, Toren Finkel⁹, Henry J Forman^{6

10}, Yvonne Janssen-Heininger¹¹, David Gems¹², Valerian E Kagan³, Balaraman Kalyanaraman¹³, Nils-Göran Larsson¹⁴, Ginger L Milne¹⁵, Thomas Nyström¹⁶, Henrik E Poulsen¹⁷, Rafael Radi¹⁸, Holly Van Remmen¹⁹, Paul T Schumacker²⁰, Paul J Thornalley²¹, Shinya Toyokuni²², Christine C Winterbourn²³, Huiyong Yin²⁴, Barry Halliwell²⁵

Affiliations

¹ MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK. mpm@mrc-mbu.cam.ac.uk.
² Department of Critical Care Medicine, Safar Center for Resuscitation Research, Children's Neuroscience Institute, University of Pittsburgh, Pittsburgh, PA, USA.
³ Department of Environmental and Occupational Health, Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA.
⁴ Federal Center of Brain Research and Neurotechnologies, Moscow, Russian Federation.
⁵ University of California, Berkeley, CA, USA.
⁶ Gerontology, Molecular & Computational Biology, and Biochemistry & Molecular Medicine, University of Southern California, Los Angeles, CA, USA.
⁷ Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark.
⁸ German Cancer Research Center, DKFZ-ZMBH Alliance and Faculty of Biosciences, Heidelberg University, Heidelberg, Germany.
⁹ University of Pittsburgh, Pittsburgh, PA, USA.
¹⁰ School of Natural Sciences, University of California, Merced, Merced, CA, USA.
¹¹ Institute of Healthy Ageing, Department of Genetics, Evolution and Environment, University College London, London, UK.
¹² University of Vermont, Burlington, VT, USA.
¹³ Medical College of Wisconsin, Milwaukee, WI, USA.
¹⁴ Division of Molecular Metabolism, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
¹⁵ Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
¹⁶ University of Gothenburg, Gothenburg, Sweden.
¹⁷ University Hospital Copenhagen, Copenhagen, Denmark.
¹⁸ Universidad de la República, Montevideo, Uruguay.
¹⁹ Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.
²⁰ Northwestern University, Evanston, IL, USA.
²¹ Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar.
²² Nagoya University Graduate School of Medicine, Nagoya, Japan.
²³ Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand.
²⁴ Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai, China.
²⁵ Department of Biochemistry and Life Sciences Institute Neurobiogy Programme, National University of Singapore, Singapore, Singapore. bchbh@nus.edu.sg.

PMID: 35760871
PMCID: PMC9711940
DOI: 10.1038/s42255-022-00591-z

Review

Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo

Michael P Murphy et al. Nat Metab. 2022 Jun.

. 2022 Jun;4(6):651-662.

doi: 10.1038/s42255-022-00591-z. Epub 2022 Jun 27.

Authors

Affiliations

¹ MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK. mpm@mrc-mbu.cam.ac.uk.
² Department of Critical Care Medicine, Safar Center for Resuscitation Research, Children's Neuroscience Institute, University of Pittsburgh, Pittsburgh, PA, USA.
³ Department of Environmental and Occupational Health, Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA.
⁴ Federal Center of Brain Research and Neurotechnologies, Moscow, Russian Federation.
⁵ University of California, Berkeley, CA, USA.
⁶ Gerontology, Molecular & Computational Biology, and Biochemistry & Molecular Medicine, University of Southern California, Los Angeles, CA, USA.
⁷ Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark.
⁸ German Cancer Research Center, DKFZ-ZMBH Alliance and Faculty of Biosciences, Heidelberg University, Heidelberg, Germany.
⁹ University of Pittsburgh, Pittsburgh, PA, USA.
¹⁰ School of Natural Sciences, University of California, Merced, Merced, CA, USA.
¹¹ Institute of Healthy Ageing, Department of Genetics, Evolution and Environment, University College London, London, UK.
¹² University of Vermont, Burlington, VT, USA.
¹³ Medical College of Wisconsin, Milwaukee, WI, USA.
¹⁴ Division of Molecular Metabolism, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
¹⁵ Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
¹⁶ University of Gothenburg, Gothenburg, Sweden.
¹⁷ University Hospital Copenhagen, Copenhagen, Denmark.
¹⁸ Universidad de la República, Montevideo, Uruguay.
¹⁹ Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.
²⁰ Northwestern University, Evanston, IL, USA.
²¹ Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar.
²² Nagoya University Graduate School of Medicine, Nagoya, Japan.
²³ Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand.
²⁴ Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai, China.
²⁵ Department of Biochemistry and Life Sciences Institute Neurobiogy Programme, National University of Singapore, Singapore, Singapore. bchbh@nus.edu.sg.

PMID: 35760871
PMCID: PMC9711940
DOI: 10.1038/s42255-022-00591-z

Abstract

Multiple roles of reactive oxygen species (ROS) and their consequences for health and disease are emerging throughout biological sciences. This development has led researchers unfamiliar with the complexities of ROS and their reactions to employ commercial kits and probes to measure ROS and oxidative damage inappropriately, treating ROS (a generic abbreviation) as if it were a discrete molecular entity. Unfortunately, the application and interpretation of these measurements are fraught with challenges and limitations. This can lead to misleading claims entering the literature and impeding progress, despite a well-established body of knowledge on how best to assess individual ROS, their reactions, role as signalling molecules and the oxidative damage that they can cause. In this consensus statement we illuminate problems that can arise with many commonly used approaches for measurement of ROS and oxidative damage, and propose guidelines for best practice. We hope that these strategies will be useful to those who find their research requiring assessment of ROS, oxidative damage and redox signalling in cells and in vivo.

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Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

**Fig. 1 |**
Summary of recommendations for measurement of ROS. Here we have summarized and abbreviated the recommendations for best practices developed in this manuscript.

See this image and copyright information in PMC

References

1. Halliwell B & Gutteridge JMC Free Radicals in Biology and Medicine 5th edn (Oxford Univ. Press, 2015).
1. Sies H et al. Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. Nat. Rev. Mol. Cell Biol 10.1038/s41580-022-00456-z (2022). - DOI - PubMed
1. Forman HJ & Zhang H Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nat. Rev. Drug Discov 20, 689–709 (2021). - PMC - PubMed
1. Lennicke C & Cochemé HM Redox metabolism: ROS as specific molecular regulators of cell signaling and function. Mol. Cell 81, 3691–3707 (2021). - PubMed
1. Winterbourn CC The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells. Biochim. Biophys. Acta 1840, 730–738 (2014). - PubMed

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Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo

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Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo

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Abstract

Conflict of interest statement

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