Fluo-4 AM (CAS: 273221-67-3) is a fluorescent calcium indicator widely utilized in biomedical research for intracellular calcium concentration measurement. This acetoxymethyl ester derivative readily diffuses across cell membranes and is hydrolyzed intracellularly by esterases, generating the calcium-sensitive fluorescent dye Fluo-4. Binding of Fluo-4 to cytosolic Ca2? ions induces a significant increase in fluorescence intensity, enabling precise real-time calcium monitoring. Structurally derived from Fluo-3 AM by substituting chlorine with fluorine, Fluo-4 AM exhibits quicker loading kinetics and approximately doubled fluorescence intensity upon excitation at 488 nm. It finds extensive application in cell signaling, functional assays, and pharmacological assessments of calcium-dependent processes.