Introduction

Generation of reactive oxygen species (ROS) at a controlled level is a feature of live cell function. Under conditions of oxidative stress, ROS production is dramatically increased, resulting in damage of membrane lipids, proteins, and nucleic acids. Oxidative damage is associated with aging as well as many diseases. Probes for measuring ROS provide important tools for study oxidative stress related diseases.

The ROS assay kit is specifically designed to detect ROS in live cells. This kit uses a unique fluorescent probe H2DCFDA to detect reactive oxygen species. H₂DCFDA is chemically reduced and acetylated form of 2′,7′-dichloro- fluorescein (DCF) and is nonfluorescent and cell-permeant. In addition, the kit provides the common inducer of ROS production tert-butyl hydroperoxide (TBHP) as a positive control.

Once H₂DCFDA enters into cells, the acetate groups are removed by intracellular esterases to form H₂DCF. H2DCF is well retained inside cells. Oxidation of H₂DCF by reactive oxygen species yields fluorescent DCF. Reactive oxygen species can be detected by monitoring the increase in fluorescence with a flow cytometer, fluorometer, microplate reader, or fluorescence microscope, using excitation sources and filters appropriate for fluorescein (FITC). Because the dyes are susceptible to photo-oxidation, low light conditions should be used for fluorescence microscopy applications whenever possible