Principle of the Procedure

The ROS Detection Kit utilizes the fluorescent probe H₂DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) for the measurement of intracellular reactive oxygen species (ROS).

H₂DCFH-DA is a non-fluorescent, cell-permeable compound that readily diffuses across cell membranes. Once inside the cell, intracellular esterases hydrolyze H₂DCFH-DA to generate DCFH, which is membrane-impermeable and therefore retained within the cells. Intracellular ROS oxidize non-fluorescent DCFH to form DCF (2′,7′-dichlorofluorescein), a highly fluorescent molecule. The fluorescence intensity of DCF is directly proportional to the intracellular ROS level.

By monitoring DCF fluorescence in live cells, researchers can effectively evaluate intracellular ROS production and oxidative stress status. DCF fluorescence can be readily detected and analyzed using flow cytometry, fluorescence microscopy, or a fluorescence microplate reader, making this one of the most established and reliable methods for ROS detection in live cells and tissues.

To facilitate assay validation and optimization, the kit includes Rosup, a positive control reagent for ROS induction. Rosup is supplied as a proprietary mixture at a concentration of 50 mg/mL and effectively stimulates intracellular ROS generation. By comparing fluorescence signals between treated and control samples, the actual ROS level can be accurately assessed.