Xcellence pro software

Cells were incubated with Fura-2 acetoxymethyl ester for 30 min at 37 °C. For recordings, 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) were perfused by gravity via a multi—barreled pipette tip. 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). The TRPV4 inhibitor HC067047 (10 µM) was perfused prior to LPS applications, as indicated in the Figures. The TLR4 inhibitor Cli-095 (1 µM) was added to the cells 20 min before measurements, and constant perfusion of this compound was kept during the measurements. PMB (300 µg ml⁻¹) was added to LPS solutions and mixed for 30 min.
For simultaneous Ca²⁺ and NO imaging, cells were incubated with Fura-2 acetoxymethyl ester as detailed above and were also pre-loaded with 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM) diacetate for 20 min. DAF-FM images were acquired using the excitation/emission wavelengths at ~488/520 nm. The fluorescence intensity of individual cells in each experiment is expressed as percentage of the average signal recorded at baseline (F/F₀, %).

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).