Medicina Molecular e Genética

An In vitro Assay to Quantify Nitrosative Component of Oxidative Stress

Abstract

S Balaiya and KV Chalam

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.