The fluorescent probe used was Fura-2 acetoxymethyl ester (AM) purchased from Invitrogen. The AM group which is lipophilic enables the hydrophilic Fura-2 to get across the plasma membrane into the cytosol. The esterases in the cytosol cleave the AM group and set the Fura-2 free in the cytoplasm. The Fura-2 binds to the Ca²⁺ and changes its own fluorescent properties as they are excited by the two wavelengths (340nm and 380nm) and emits at 510 nm (figure). By the ratiometric method the amount of Ca²⁺ bound to the Fura-2 can be measured.

Figure: Excitation spectrum of Fura-2 dye measured at emission wavelength of 510nm. Two excitation peaks were seen around 340nm and 380nm wavelength. The excitation peak for 340nm increases with increase in Ca²⁺ concentration, and the excitation peak for 380nm decreases with increase in the Ca²⁺ concentration