title: Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses.
abstract: Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Jacek
authors.initials: J
authors.lastname: Zielonka
authors.forename: Monika
authors.initials: M
authors.lastname: Zielonka
authors.forename: Adam
authors.initials: A
authors.lastname: Sikora
authors.forename: Jan
authors.initials: J
authors.lastname: Adamus
authors.forename: Joy
authors.initials: J
authors.lastname: Joseph
authors.forename: Micael
authors.initials: M
authors.lastname: Hardy
authors.forename: Olivier
authors.initials: O
authors.lastname: Ouari
authors.forename: Brian P
authors.initials: BP
authors.lastname: Dranka
authors.forename: Balaraman
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D000818:Animals; D002460:Cell Line; D051379:Mice; D008954:Models, Biological; D015335:Molecular Probes / Q000737:chemistry / Q000494:pharmacology; D010084:Oxidation-Reduction; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D015335:Molecular Probes; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species