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Measurement of Oxidative Stress Markers In Vitro Using Commercially Available Kits

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Measuring Oxidants and Oxidative Stress in Biological Systems

Part of the book series: Biological Magnetic Resonance ((BIMR,volume 34))

Abstract

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are highly reactive molecules, with significant effects in human diseases including cancer and cardiovascular disease. The ability to accurately and precisely detect the formation of free radicals within cells and tissues is crucial to developing proper treatments for the problems caused by ROS/RNS. Fluorescent probes have become widely available reagents of detecting ROS/RNS within cells. Several commercially available kits have shown their specificity toward detecting the formation of ROS and RNS. In this chapter, we discuss the principle behind each kit and the benefits and shortcomings of these kits, namely dihydroethidium (DHE), dichlorohydrofluoresin diacetate (DCF-DA), 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM diacetate), and 10-acetyl-3,7-dihyrdroxyphenoxazine (Amplex red). DHE is used to specifically detect superoxide, while DCF-DA readily detects hydroxyl radicals. Amplex red is used to detect hydrogen peroxide, and DAF-FM is used for measuring nitric oxide. However, due to the nature of their reactivity, the probes are not absolutely specific for the noted ROS/RNS species, and will react with others. ROS measurement may need to be made in real time, and they are short-lived within the cell, especially superoxide and nitric oxide. This chapter explains the mechanism behind each chemical kit, the protocols used with the kit, and show typical results after imaging. Additionally, an assessment is made on the use of the kit, identifying the advantages and disadvantages of each probe.

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Acknowledgement

I would like to acknowledge funding from National Institutes of Health (NIH) R01 Grant HL136232 to MK and HL133050 to HS.

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Authors and Affiliations

  1. Department of Emergency Medicine, Dorothy M. Davis Heart Lung and Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH, USA

    Bryan Gardiner, Julie A. Dougherty, Mark Angelos, Chun-An Chen & Mahmood Khan

  2. Department of Physiology and Cell Biology, The Ohio State University Wexner Medical Center, Columbus, OH, USA

    Devasena Ponnalagu, Harpreet Singh & Mahmood Khan

Authors
  1. Bryan Gardiner
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  2. Julie A. Dougherty
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  3. Devasena Ponnalagu
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  4. Harpreet Singh
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  5. Mark Angelos
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  6. Chun-An Chen
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  7. Mahmood Khan
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Corresponding author

Correspondence to Mahmood Khan .

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Editors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Denver, Centennial, CO, USA

    Lawrence J. Berliner

  2. Department of Internal Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, Ohio State University College of Medicine, Dorothy M. Davis Heart and Lung Research Institute, Columbus, OH, USA

    Narasimham L. Parinandi

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Gardiner, B. et al. (2020). Measurement of Oxidative Stress Markers In Vitro Using Commercially Available Kits. In: Berliner, L., Parinandi, N. (eds) Measuring Oxidants and Oxidative Stress in Biological Systems. Biological Magnetic Resonance, vol 34. Springer, Cham. https://doi.org/10.1007/978-3-030-47318-1_4

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