Fluo-3FF AM is a low-affinity (Kd = 42 μM), Mg 2+ insensitive, and relatively photostable fluorescent Ca 2+ sensitive indicator (Abs/Em = 462 nm/526 nm). It is essentially non-fluorescent but exhibits strong fluorescence enhancement upon entry into cells and binding to calcium. [1]

Fluo-3FF AM working solution preparation: 1.1 To prepare the stock solution, dissolve Fluo-3FF AM in 0.025% (w/v) Pluronic F-127/dimethyl sulfoxide to obtain a 1 mM stock solution. Note: It is recommended to aliquot and store Fluo-3FF AM stock solution at -20℃ or -80℃, protected from light. 1.2 For the working solution, dilute in balanced buffer to achieve a final concentration of 5 μM Fluo-3 AM. Note: Adjust the concentration of Fluo-3FF AM working solution as needed and prepare fresh for use. Cell staining: 2.1 Culture adherent cells on sterile coverslips. 2.2 Remove the coverslip from the culture medium and aspirate excess medium. 2.3 Add 100 μL of dye working solution, gently swirl to fully cover the cells, and incubate for 60 minutes. 2.4 Aspirate the dye working solution and wash with PSS at 4°C for 60 minutes.