4.4. Ca2+ Imaging with Fura-2-AM

Measurements of intracellular Ca²⁺ ([Ca²⁺]_i) were performed using the fluorescent Ca²⁺ indicator fura-2 AM. Cells were loaded with 5 μM of fura-2 AM dissolved in extracellular solution with 0.1% pluronic acid to improve dye uptake, for 45 min at 37 °C. Cell coverslips were placed on the stage of an inverted Nikon TE200 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a dual-excitation fluorometric Ca²⁺ imaging system (Hamamatsu, 325-6, Sunayama-cho, Naka-ku, Hamamatsu City, Shizuoka, Japan). Cells were excited at 340 and 380 nm, at a sampling rate of 0.5 Hz, and fluorescence emission, measured at 510 nm, was acquired with a digital CCD camera (Hamamatsu C4742-95-12ER). The external solutions were the same as used in the patch-clamp experiments. The fluorescence ratio F340/F380 was used to monitor [Ca²⁺]_i changes. Monochromator settings, chopper frequency, and data acquisition were controlled with dedicated software (Aquacosmos/Ratio U7501-01, Hamamatsu, Japan). [Ca²⁺]_i was calculated according to the method of Grynkiewicz et al. [96], using a KD of 140 nM for the Ca²⁺/fura-2 complex. Data were analyzed using SigmaPlot 8.0 software. Results are presented as mean ± standard error of different cells from at least 4 independent experiments.