Ix2 ucb control box

[Ca²⁺]_i measurements were performed as previously described^{17 (link),20 (link)}. Briefly, after being loaded with Fura-2-AM, HK-2 cells were placed on an IX81 motorized inverted microscope equipped with an IX2-UCB control box (Olympus USA, Center Valley, PA). Experiments were performed in a microincubator set at 37 °C with a gas mixture of 5% CO₂/95% air. Cells were immersed in Ca²⁺-free external solution. Ratiometric (340/380) measurements of [Ca²⁺]_i were recorded using digital microscopy imaging software (SlideBook version 5.0, 3i, Intelligent Imaging Innovations, Denver, CO). Fura-2 fluorescence was recorded at an emission peak absorbance at 500 nm wavelength with excitation peak absorbance that alternated between wavelengths of 340 and 380 nm. Time-lapsed measurements were set to 800 time points at a 1 s interval. 50–150 cells were individually selected as regions of interest (background fluorescence automatically subtracted prior to 340/380 ratio calculation and graphing). Capture analyses were performed offline using Slidebook^™ software and further quantitated with statistical analysis using Origin 6.1.

Ratiometric [Ca²⁺]_i measurements were conducted as previously described [3 (link),52 ]. Cells were loaded with Fura-2AM and placed on an IX81 motorized inverted microscope equipped with an IX2-UCB control box (Olympus USA, Center Valley, PA). Measurements were performed in a humidified microincubator with gas mixture of 95% air and 5% CO₂ at constant temperature set to 37°C. Fura-2 fluorescence was recorded at emission peak absorbance of 500 nm wavelength with excitation peak absorbance that continuously shifted 340 nm and 380 nm wavelengths. To obtain the time-dependent changes, time-lapse recordings were set at different time intervals (300–500 s) for taking images at 1 s intervals, and then averaged the recording data from at least 50–150 cells to select the region of interest, whereas the background fluorescence was automatically subtracted prior obtaining 340/380 ratio calculation and graphing. Analysis was performed offline using Slidebook™ software and further analyzed using statistical analysis by Origin 6.1.

Fura-2 [Ca²⁺]_i measurements were performed as previously described [30 (link),49 ]. Briefly, after being loaded with Fura-2, PT cells were placed on an IX81 motorized inverted microscope equipped with an IX2-UCB control box (Olympus USA, Center Valley, PA). Experiments were performed in a microincubator set at 37 °C with a gas mixture of 5% CO₂/95% air. Cells were immersed in Ca²⁺-free external solution. Ratiometric (340/380) measurements of [Ca²⁺]_i were recorded using digital microscopy imaging software (SlideBook version 5.0, 3i, Intelligent Imaging Innovations, Denver, CO). Fura-2 fluorescence was recorded at an emission peak absorbance at 500 nm wavelength with excitation peak absorbance that alternated between wavelengths of 340 and 380 nm. Time-lapsed measurements were set to 800 time points at a 1 s interval. 50–150 cells were individually selected as regions of interest (background fluorescence automatically subtracted prior to 340/380 ratio calculation and graphing). Capture analyses were performed offline using Slidebook™ software and further quantitated with statistical analysis using Origin 6.1.