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).