title: Rapid and specific measurements of superoxide using fluorescence spectroscopy.
abstract: Superoxide plays a key role in many pathological processes; however, detection of superoxide by one of the most common methods using dihydroethidium (DHE) may be unspecific because of overlapping fluorescence of the superoxide-specific product, 2-OH-ethidium (2OH-E), and the unspecific oxidation product, ethidium. Here, we show a new optimized fluorescence spectroscopy protocol that allows rapid and specific detection of superoxide in cell-free systems and intact cells using DHE. We defined new optimized fluorescent settings to measure the superoxide-specific product and minimize the interference of unspecific DHE oxidation products. Using this protocol, we studied real-time superoxide production by xanthine oxidase- and menadione-treated cultured cells. Specificity of the plate reader-based superoxide measurements was confirmed by the inhibition of fluorescence with superoxide dismutase and high-performance liquid chromatography (HPLC) analysis. We show that limitations of the HPLC-based analysis can be overcome by the optimized fluorescence spectroscopy.
authors.affiliation: Vanderbilt University Medical Center, Nashville, TN 37212, USA. r.nazar@Vanderbilt.edu
authors.forename: Rafal R
authors.initials: RR
authors.lastname: Nazarewicz
authors.forename: Alfiya
authors.initials: A
authors.lastname: Bikineyeva
authors.forename: Sergey I
authors.initials: SI
authors.lastname: Dikalov
mesh_terms: D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D004996:Ethidium / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013481:Superoxides / Q000032:analysis*; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: C067883:dihydroethidium; D013481:Superoxides; D014969:Xanthine Oxidase; D004996:Ethidium