Stage top cage incubator

Cells were grown on glass-bottom plates. After treatment with the appropriate drug, the cells were incubated with 5 µM Fura-2 AM (TEFLabs, Austin, TX) at 37 °C for 30 minutes. After washing with DPBS, the cells were observed under a 40 × objective lens with a Nikon Eclipse Ti-E microscope. Fura-2 fluorescence images at excitation of 340/380 nm and emission of 510 nm were recorded. After equilibration under the microscope for 20 minutes, baseline calcium was recorded for 2 minutes and experimental data were acquired. Fluid-shear stress was then applied to cells with an Instech P720 peristaltic pump. The perfused fluid was pumped into the cell culture dish and retained at a shear stress of 1 dyne/cm² (for epithelial cells) or 8 dyne/cm² (for endothelial cells) with a constant flow rate of about 20 or 160 μl/sec, respectively. At the end of each experiment, the maximum calcium signal was obtained by perfusion of ATP (10 µM) to confirm cell viability. In addition to autofluorescence, both minimum and maximum fluorescence was collected as previously described^{84 (link)}. Conditions for all experiments were maintained at 37 °C and 5% CO₂ in a stage top cage incubator (okoLab, Burlingame, CA). Calcium analysis was then followed a standard calculation as previously described^{84 (link)}.

Cells were grown on glass-bottom plates to enable live microscopy imaging. After incubation for 20-hour without or with 1μM rapamycin, cells were loaded with 5 μM Fura2-AM (TEFLabs, Austin, TX) at 37°C for 30 min. Cells were then washed with DPBS (Dulbecco's Phosphate-Buffered Saline) and observed under a 40× objective lens with a Nikon Eclipse Ti-E microscope controlled by Elements software. Cytosolic calcium was observed by recording calcium-bound Fura excitation fluorescence at 340/380 nm and emission at 510 nm. Baseline calcium was observed for 2 minutes prior to data acquisition. Fluid-shear stress was then applied to cells utilizing an Instech P720 peristaltic pump with an inlet and outlet setup. The fluid was perfused on the glass-bottom plates at shear stress of 0.2, 0.6 or 1.0 dyne/cm² for epithelial cells and 2.0, 5.0 or 8.0 dyne/cm² for endothelial cells. After each experiment, the maximum calcium signal was obtained by perfusion of ATP (10μM) to confirm cell viability. Conditions for all experiments were maintained at 37°C and 5% CO₂ in a stage top cage incubator (okoLab, Burlingame, CA). Calcium analysis was then followed a standard calculation as previously described (Upadhyay et al., 2014 (link)).