An In vitro Assay to Quantify Nitrosative Component of Oxidative Stress
S Balaiya and KV Chalam*
Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, Florida, USA
- Corresponding Author:
- KV Chalam
Department of Ophthalmology
University of Florida College of Medicine
Jacksonville, Florida, USA
E-mail: [email protected]
Received date: April 01, 2014; Accepted date: August 07, 2014; Published date: October 20, 2014
Citation: Balaiya S, Chalam KV (2014) An In vitro Assay to Quantify Nitrosative Component of Oxidative Stress. J Mol Genet Med 8:120. doi:10.4172/1747-0862.1000120
Copyright: © 2014 Balaiya S, et al., This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Oxidative stress is a major contributing factor in a variety of neurodegenerative and vascular diseases. In vitro assessment of a major oxidant reactive nitrogen oxide species (RNOS) using dihydrorhodamine 123 (DHR 123) is a useful assay to quantify the reactive oxygen species in a cell. DHR 123, non-fluorescent laser dye freely penetrates the cell membrane and stains the mitochondria. Density of staining varies with the level of peroxynitrite (O=NOO-); as a result of interaction of superoxide anion (O2-) and nitric oxide (NO). The fluorescence is read using a spectrophotometer. Cells are seeded in 24 or 96- well plate and DHR 123 working solution is added after appropriate treatment. The fluorescence is read after 60 minutes of incubation at 485/528 nm with spectrophotometer. This assay is more sensitive and forms a stable end product than comparable assays and takes 90 minutes to complete.