Methods for Detection of NOX-Derived Superoxide Radical Anion and Hydrogen Peroxide in Cells
NADPH oxidases (NOX) are transmembrane enzymes, which catalyze the formation of reactive oxygen species (ROS). In humans and most mammals, the NOX family comprises seven members, namely, NOX1-5 and the dual oxidases DUOX1 and 2. The primary product of most NOX isoforms is the superoxide radical anion O2ċ–, which is rapidly dismutated in hydrogen peroxide (H2O2), while NOX4 and DUOX mostly generate H2O2. ROS are multifunctional molecules in tissues, and NOX-derived ROS cellular functions are as diverse as microbial killing (NOX2), thyroid hormone synthesis (DUOX2), or otoconia formation in the inner ear (NOX3). NOX are potential pharmacological targets in numerous diseases such as diabetes, fibrosis, and brain ischemia, and NOX inhibitors are currently under development. Here we describe two cellular assays to detect extracellular O2ċ– and H2O2 in cells overexpressing specific NOX isoforms and their subunits.
Key wordsNADPH oxidase Hydrogen peroxide Superoxide radical anion Peroxidase Amplex red WST-1 Cellular assay
This work was supported by the European Community’s Framework Programme (FP7/2007–2013) under grant agreement number 278611.
- 5.Maghzal GJ, Krause KH, Stocker R, Jaquet V (2012) Detection of reactive oxygen species derived from the family of NOX NADPH oxidases. Free Radic Biol Med 53(10):1903–1918. https://doi.org/10.1016/j.freeradbiomed.2012.09.002 CrossRefPubMedGoogle Scholar
- 6.Seredenina T, Chiriano G, Filippova A, Nayernia Z, Mahiout Z, Fioraso-Cartier L, Plastre O, Scapozza L, Krause KH, Jaquet V (2015) A subset of N-substituted phenothiazines inhibits NADPH oxidases. Free Radic Biol Med 86:239–249. https://doi.org/10.1016/j.freeradbiomed.2015.05.023 CrossRefPubMedGoogle Scholar
- 7.Tan AS, Berridge MV (2000) Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents. J Immunol Methods 238(1–2):59–68CrossRefGoogle Scholar