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HPLC-Based Monitoring of Oxidation of Hydroethidine for the Detection of NADPH Oxidase-Derived Superoxide Radical Anion

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Book cover NADPH Oxidases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1982))

Abstract

Hydroethidine is a fluorogenic probe that in the presence of the superoxide radical anion is oxidized to a red fluorescent product, 2-hydroxyethidium. In cells, hydroethidine is also oxidized to other products, including red fluorescent ethidium. Thus, selective monitoring of 2-hydroxyethidium is required for specific detection of the superoxide radical anion. Here, we provide protocols for HPLC- and LC-MS-based quantitation of 2-hydroxyethidium, among other oxidation products. Also, a protocol for continuous sampling for real-time monitoring of superoxide production using rapid HPLC measurements of 2-hydroxyethidium is described.

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Acknowledgment

This work was supported by NIH grants NCI U01 CA178960 and R01 AA022986 to B.K.

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Authors and Affiliations

  1. Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA

    Jacek Zielonka, Monika Zielonka & Balaraman Kalyanaraman

  2. Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI, USA

    Jacek Zielonka, Monika Zielonka & Balaraman Kalyanaraman

  3. Cancer Center, Medical College of Wisconsin, Milwaukee, WI, USA

    Jacek Zielonka & Balaraman Kalyanaraman

Authors
  1. Jacek Zielonka
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  2. Monika Zielonka
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  3. Balaraman Kalyanaraman
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Corresponding author

Correspondence to Jacek Zielonka .

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Editors and Affiliations

  1. Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland

    Ulla G. Knaus

  2. Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Thomas L. Leto

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Zielonka, J., Zielonka, M., Kalyanaraman, B. (2019). HPLC-Based Monitoring of Oxidation of Hydroethidine for the Detection of NADPH Oxidase-Derived Superoxide Radical Anion. In: Knaus, U., Leto, T. (eds) NADPH Oxidases. Methods in Molecular Biology, vol 1982. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9424-3_14

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  • DOI: https://doi.org/10.1007/978-1-4939-9424-3_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9423-6

  • Online ISBN: 978-1-4939-9424-3

  • eBook Packages: Springer Protocols

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