Fluorescent Ca2+ Imaging of Endothelial Cells

Arteries were incubated in a loading solution containing a fluorescent Ca²⁺ indicator, Cal-520 acetoxymethyl ester (Cal-520/AM; 5 µM), 0.02% Pluronic F-127, and 0.35% dimethyl sulfoxide in PSS for 30 min (37 °C). All Ca²⁺ measurements were carried out in PSS. Cal-520 was excited with 488-nm wide‐field epifluorescence illumination provided by a light-emitting diode illumination system (PE-300Ultra, CoolLED) and imaged using an EMCCD camera (13-µm pixel size iXon Life 888; Andor), through a 16× (water immersion; numerical aperture of 0.8; Nikon CFI75) objective lens or 40× (water immersion; numerical aperture of 0.8; Nikon CFI Apo) objective lens. Fluorescence emission was recorded at 10 Hz. Fluorescence illumination was controlled, and images (16-bit depth) were captured by µManager.
Ca²⁺ imaging recordings were analyzed using custom FIJI macros and custom analysis software written in the Python 2.7 programming language (5 (link), 24 (link)). ROIs were automatically generated for each endothelial cell so that a direct comparison of the Ca²⁺ activity in each cell between various pharmacological applications could be made (24 (link), 47 (link)).