Glass bottomed dishes

HLMVECs grown on glass-bottomed dishes (Becton Dickinson) were loaded with fura-2 AM (3 μM, Life Technologies) for 20 min at 37°C in culture medium without supplements. The medium was then replaced with medium comprising: 150 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 5.6 mM glucose, and 25 mM HEPES (pH 7.4), and, after ∼10 min, cells were used for experiments at 25°C. Fura-2 fluorescence was excited at 340 and 380 nm and collected at 510 ± 80 nm using an Axiovert 100 inverted microscope (Carl Zeiss) equipped with Plan-Apo 60× with the numerical aperture (NA) 1.4 oil immersion objective, Lambda DG-4 switcher illumination system (Sutter Instruments), AxioCom Hsm camera (Zeiss), fura-2 filter set (Chroma), and AxioVision Physiology Acquisition module. Images were collected at 2-s intervals. Fluorescence ratios (F₃₄₀/F₃₈₀) were calculated within a circular region of interest (radius 3 μm) for each cell after subtraction of intracellular background fluorescence, determined by quenching fura-2 fluorescence by addition of 3 μM ionomycin with 5 mM MnCl₂. [Ca²⁺]_c was calculated from F₃₄₀/F₃₈₀ ratios by reference to Ca²⁺ standard solutions (Life Technologies). Measurements of cytosolic IP₃ concentrations are described in Supplemental Experimental Procedures.