The production of reactive oxygen species, including H2O2, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H2O2 release from cells (Panus et al., 1993; Murthy et al., 2010; Larson-Casey et al., 2016; Larson-Casey et al., 2014; He et al., 2011). This assay takes advantage of H2O2-mediated oxidation of horseradish peroxidase (HRP) to Complex I, which, in turn, oxidizes p-hydroxyphenylacetic acid (pHPA) to a stable, fluorescent pHPA dimer (2,2'-dihydroxy-biphenyl-5,5’ diacetate [(pHPA)2]). The H2O2-dependent HRP-mediated oxidation of pHPA is a sensitive and specific assay for quantifying H2O2 release from cells. This assay can measure H2O2 release from whole cells, mitochondria, or the NADPH oxidase.
[Background] H2O2 generation primarily results from dismutation of superoxide anion (O2-), which occurs at a rapid rate (105-106 M-1 s-1) non-enzymatically. Unlike O2-, H2O2 can traverse membranes easily, so it is able to oxidize multiple molecules. ROS can be toxic to cells by oxidizing proteins, lipids, and nucleic acids and are associated with many human diseases. This protocol allows for detection of H2O2 release from NADPH oxidase or mitochondria in various cell types.
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