Widely used in cell biology for viability and permeability assays, this compound serves as a fluorescent indicator for intracellular esterase activity and membrane integrity. It easily penetrates live cells, where nonspecific intracellular esterases cleave the AM (acetoxymethyl) ester groups, trapping the fluorescent calcein anion inside. The de-esterified product emits bright green fluorescence, making it ideal for real-time visualization of live cells in microscopy and flow cytometry. Commonly applied in cytotoxicity studies, it helps distinguish live cells from dead ones in co-culture systems. It is also used in cell migration and invasion assays, as well as in high-throughput screening to assess cell health. Due to its strong fluorescence and low toxicity, it is frequently combined with other probes like propidium iodide or ethidium homodimer to enable multiplex staining. Additionally, it supports research in drug delivery, nanotoxicology, and stem cell tracking.