Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

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

12/06/2011

Pubmed ID

22139901

Pubmed Central ID

PMC3270955

DOI

10.1074/jbc.M111.309062

Scopus ID

2-s2.0-84856248241 (requires institutional sign-in at Scopus site)   163 Citations

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.

Author List

Zielonka J, Zielonka M, Sikora A, Adamus J, Joseph J, Hardy M, Ouari O, Dranka BP, Kalyanaraman B

Authors

Micael Joel Hardy PhD Visiting Assistant Professor in the Biophysics department at Medical College of Wisconsin
Balaraman 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

Animals
Cell Line
Mice
Models, Biological
Molecular Probes
Oxidation-Reduction
Reactive Nitrogen Species
Reactive Oxygen Species