4.4. Measurement of [Ca2+]i Concentrations

Hep3B cells were trypsin-digested, allowed to sediment, resuspended in Hepes-buffered medium (HBM), consisting of 20 mM Hepes (pH 7.4), 103 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 0.5 mM CaCl₂, 25 mM NaHCO₃, 15 mM glucose, and 0.1% bovine serum albumin (fatty acid free), and then incubated for 40 min with 5 μM of Fura 2-AM. [Ca²⁺]_i levels were estimated by measuring changes in Fura 2 fluorescence using an emission wavelength of 510 nm and excitation wavelengths of 340 nm and 380 nm every 0.1 s using a F4500 fluorescence spectrophotometer (Hitachi, Japan). The ratios of fluorescence intensities (λ340/λ380) at these two wavelengths were used as a surrogate of [Ca²⁺]_i, as previously described [18].