Fluo-8,AM is one of the most commonly used fluorescent probes to detect intracellular calcium concentration.Fluo-8, which penetrates the cell membrane and enters the cell, is cut by the intracellular esterase to form fluO-8, and thus is trapped in the cell. Fluo-8 is almost non-fluorescent when it exists in the form of free ligand, but when it combines with intracellular calcium ions, it can produce strong fluorescence, with the maximum excitation wavelength of 490nm and the maximum emission wavelength of 514nM.

Load Cells with Fluo-8? AM Esters: Prepare a 2 to 5 mM stock solution of Fluo-8? AM esters in high-quality, anhydrous DMSO. Prepare a working solution of 1 to 10 μM in Hanks and Hepes buffer (HHBS). For most of cell lines, Fluo-8? reagents with a concentration ranging from 4-5 uM are recommended. Probenecid (1–2.5 mM) or sulfinpyrazone (0.1–0.25 mM) may be added to the cell medium to reduce leakage of the de-esterified indicators. Add equal volume of the dye working solution into your cell plate. Incubate the dye-loading plate at a cell incubator (20-60minutes). Replace the dye working solution with HHBS to remove excess probes. Run the experiments at Ex/Em = 490/525 nm.