2,7-Dichlorodihydrofluorescein diacetate

2,7-Dichlorodihydrofluorescein diacetate (CAS No.: 4091-99-0) is a widely used cell-permeable fluorogenic probe for the detection of intracellular reactive oxygen species (ROS) in biomedical research and drug discovery. Classified as a nonfluorescent diacetate derivative of dichlorofluorescin, this compound readily diffuses across cellular membranes and is subsequently deacetylated by intracellular esterases to yield the nonfluorescent intermediate dichlorodihydrofluorescein, which becomes trapped within cells. Upon oxidation by ROS and related reactive nitrogen species, particularly strong oxidants such as peroxynitrite, this intermediate is converted into the highly fluorescent dichlorofluorescein, enabling sensitive detection of oxidative stress through fluorescence-based methods. The resulting green fluorescence intensity, typically measured with excitation and emission maxima in the approximate range of 485–502 nm and 523–527 nm, correlates with intracellular ROS levels and can be quantified using fluorescence microscopy, flow cytometry, or plate-based assays. Although not specific to a single molecular target or pathway, this probe is broadly applied to monitor redox homeostasis, oxidative damage, and signaling events associated with mitochondrial dysfunction, inflammation, and cytotoxicity. In vitro applications commonly involve cell-based assays evaluating oxidative stress induced by chemical agents, nanoparticles, or drug candidates, while experimental concentrations generally fall within the micromolar range depending on assay conditions and cell type. Due to known limitations in specificity and potential artifacts arising from probe oxidation mechanisms, careful experimental design and appropriate controls are essential when interpreting results in mechanistic and translational studies.