Fluo-4 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 live cells. Once inside, cellular esterases remove the AM group, trapping the active dye within the cytoplasm. When bound to calcium ions, Fluo-4 exhibits a strong increase in fluorescence, making it ideal for real-time monitoring of calcium signaling. It is commonly used in fluorescence microscopy, flow cytometry, and plate reader assays. Applications include studying neuronal activity, muscle contraction, immune cell activation, and drug effects on cellular signaling pathways. Its high sensitivity and compatibility with standard FITC filter sets make it a preferred choice in many life science experiments.