Mt 10 illumination system

Urothelial cells were incubated with Fura-2 acetoxymethyl (2 μM) ester for 30 min at 37°C. During recordings cells were perfused by gravity via a multi-barreled pipette tip with bath solutions prepared in Krebs, containing (in mM): 150 NaCl, 6 KCl, 1.5 CaCl₂, 1 MgCl₂, and 10 HEPES, 10 glucose and titrated to 7.4 with NaOH. Intracellular Ca²⁺ concentration was monitored through the ratio of fluorescence measured upon alternating illumination at 340 and 380 nm using an MT-10 illumination system and the Xcellence pro software (Olympus Belgium N.V., Berchem, Belgium).

For intracellular Ca²⁺ imaging experiments cells were incubated with 2 µM Fura-2 AM (Biotium, Hayward, CA, USA) for 40 min at 37 °C in a humidity-controlled incubator. Fluorescence was measured with alternating excitation at 340 and 380 nm using a monochromator-based imaging system consisting of an MT-10 illumination system (Tokyo, Japan) and Cell^M software from Olympus. All experiments were performed using the standard Krebs solution (see above) at 25 °C. Fluorescence intensities were corrected for background signal and presented as the ratio F340/F380 from which intracellular Ca²⁺ concentration was calculated as described previously [77 ]. Data were analyzed and presented as mean ± s.e.m. using Origin 9.0 (OriginLab Corporation).

For intracellular Ca²⁺ measurements, cells were incubated for 30min with 2µM Fura-2 acetoxymethyl (AM) ester (Biotium, Hayward, CA, USA) in cell culture medium. Fluorescent signals were evoked during alternating illumination at 340 and 380 nm using an MT-10 illumination system (Tokyo, Japan) and CellM software from Olympus. Absolute Ca²⁺ concentrations were calculated from the ratio of these fluorescent signals as described before [39 (link)]. Experiments were performed at room temperature with standard extracellular perfusion buffer containing (in mM) 150 NaCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, adjusted to pH 7.4 using NaOH. For stimulation of the cells, the following modulators were used: GSK1016790A (10 nM), 4αPDD (10 µM), arachidonic acid (10 µM), and ionomycin (1 µM), all from Sigma-Aldrich (Bornem, Belgium). In experiments where osmotic responses were analyzed, we used a modified isotonic solution containing (in mM): 105 NaCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 90 mannitol, adjusted to pH 7.4 using NaOH. A hypotonic solution was obtained by omitting mannitol from the modified isotonic solution.