Fluo-4 AM is a cell-permeable calcium ion (Ca²⁺) fluorescent indicator/chemical probe suitable for fluorescence imaging, flow cytometry, and plate screening, with an excitation wavelength of approximately 488 nm and an emission wavelength of 512–520 nm.

Fluo-4 AM stock solution was prepared by dissolving it in DMSO at a concentration of 1 mM, and was used immediately to maintain activity. The working solution was freshly diluted to 1 μM using HHBS buffer containing 0.04% Poloxamer 407 (F127), protected from light. Approximately 1×10⁶ cells were collected per sample by centrifugation at 3000×g for 5 minutes at 4°C and washed once with PBS. The cell pellet was resuspended in 0.5 mL PBS, and 0.5 μL of Fluo-4 AM was added to achieve a final concentration of 1 μM. Cells were incubated at 37°C for 1 hour in the dark. After incubation, cells were washed three times with PBS (3000×g, 2 minutes each) and finally resuspended in 1 mL PBS. Intracellular calcium levels were assessed by flow cytometry using excitation/emission wavelengths of 485/526 nm. [1]