Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses. J Biol Chem 2012 Jan 27;287(5):2984-95
Date
12/06/2011Pubmed ID
22139901Pubmed Central ID
PMC3270955DOI
10.1074/jbc.M111.309062Scopus ID
2-s2.0-84856248241 (requires institutional sign-in at Scopus site) 163 CitationsAbstract
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.
Author List
Zielonka J, Zielonka M, Sikora A, Adamus J, Joseph J, Hardy M, Ouari O, Dranka BP, Kalyanaraman BAuthors
Micael Joel Hardy PhD Visiting Assistant Professor in the Biophysics department at Medical College of WisconsinBalaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of Wisconsin
Jacek M. Zielonka PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
AnimalsCell Line
Mice
Models, Biological
Molecular Probes
Oxidation-Reduction
Reactive Nitrogen Species
Reactive Oxygen Species