Single-Cell Ca2+ iVSMC Imaging
iVSMCs were loaded with the Ca2+-sensitive dye Fluo-3, AM (3 μmol/L 60 min) (Thermofisher). Cells were maintained at 37°C using a Peltier unit and continually perfused with Krebs-Henseleit buffer (mmol/L: 134 NaCl, 6 KCl, 1 MgCl2, 1.2 KH2PO4, 10 glucose, 10 HEPES, 1.3 CaCl2, pH 7.4). Real-time images were taken using an epifluorescence Nikon Eclipse TE200 microscope (Nikon) (×20 objective) and Volocity 6.1.1 image software (Quorum Technologies). Cells were stimulated with vasoconstrictors applied via the perfusion line for 45 seconds, and Fluo-3 emission was assessed at ≥520 nm. [Ca2+]i changes are displayed as the fluorescence emission relative to basal fluorescence (F/F0).