2',7'-Dichlorofluorescein diacetate

2',7'-Dichlorofluorescein diacetate (CAS No.: 2044-85-1) is a widely used cell-permeable fluorogenic probe for monitoring intracellular reactive oxygen species (ROS) and overall oxidative stress in biomedical research and drug discovery. Initially nonfluorescent, this diacetate derivative readily diffuses into cells where it is rapidly deacetylated by intracellular esterases to yield a nonfluorescent intermediate that is subsequently oxidized by ROS—predominantly hydrogen peroxide and related reactive intermediates—into the highly fluorescent dichlorofluorescein, enabling sensitive detection by fluorescence microscopy, flow cytometry, or plate-based assays with characteristic green emission. Functionally, it serves as a general redox indicator rather than a selective sensor, reporting on oxidative processes downstream of multiple pathways including mitochondrial dysfunction, NADPH oxidase activity, and inflammatory signaling cascades, and it has also been used to assess nitric oxide–related oxidative chemistry under certain conditions. This probe is extensively applied in cancer biology, toxicology, and pharmacology to quantify oxidative stress in cultured cells, including models of breast and liver cancer, and to evaluate the impact of candidate therapeutics or environmental stressors on cellular redox balance. Experimental use typically involves low micromolar loading concentrations, with assay conditions and incubation times optimized according to cell type and study design, making it a versatile tool for high-content screening and mechanistic studies of oxidative injury and redox-regulated signaling.