5(6)-Carboxyfluorescein diacetate

5(6)-Carboxyfluorescein diacetate (CAS No.: 124387-19-5) is a cell-permeable fluorescent ester widely used as a viability and metabolic activity probe in biomedical research and drug discovery. Upon passive diffusion into cells, it is hydrolyzed by intracellular nonspecific esterases to yield carboxyfluorescein, a negatively charged fluorescent species that is efficiently retained within cells possessing intact plasma membranes, thereby enabling sensitive discrimination of viable cells from compromised or dead populations. This mechanism links fluorescence generation directly to esterase activity and membrane integrity, making it a functional indicator of cellular health and enzymatic activity. The probe exhibits excitation and emission maxima around 494 and 521 nm, respectively, and demonstrates relatively slow efflux kinetics, supporting sustained intracellular signal detection in assays such as flow cytometry and fluorescence microscopy. Although it does not act on a defined molecular target or signaling pathway, its readout reflects global cellular enzymatic function and membrane permeability, typically producing responses in the low micromolar concentration range depending on cell type and assay conditions. 5(6)-Carboxyfluorescein diacetate is commonly applied in studies of cell viability, cytotoxicity, metabolic profiling, and membrane integrity, as well as in high-throughput screening workflows, where experimental concentrations and incubation conditions are adjusted according to specific biological models and research objectives.