Fluo-3 AM is widely used in biological research for detecting intracellular calcium levels. It easily crosses cell membranes due to its AM (acetoxymethyl) ester group, allowing it to enter cells where esterases cleave the AM group, trapping the active dye inside. Once activated, Fluo-3 binds to free calcium ions in the cytoplasm, resulting in a strong increase in fluorescence intensity when excited by visible light, typically from an argon laser at 488 nm. This property makes Fluo-3 AM a key tool in flow cytometry, fluorescence microscopy, and plate reader assays for monitoring calcium signaling in live cells. It is commonly used in studies involving cell activation, neurotransmission, muscle contraction, and apoptosis. Its compatibility with standard FITC filter sets and laser systems makes it convenient for use in many laboratory setups. However, it does not allow ratiometric measurements, so changes in fluorescence reflect both calcium concentration and variations in dye loading or cell thickness.