4.10. Calcium measurements

Changes in intracellular Ca²⁺ levels were determined by the fluorescent Ca²⁺ -sensitive dye Fluo-4 AM ({"type":"entrez-nucleotide","attrs":{"text":"F14201","term_id":"860754","term_text":"F14201"}}F14201, Invitrogen), following published protocols [66]. MuSCs were incubated for 30 min s at 37 °C with 5 μM Fluo-4 AM in Ca²⁺ and Mg²⁺free HBSS-HEPES buffer, after which cells were washed 3 times with HBSS-HEPES buffer. Time-lapse images were acquired at every 1.5s with a Leica SP8 MP Microscope using an immersion 40× objective. Excitation wavelength was set to 488 nm, and emission was recorded at 535 ± 15 nm. Analysis and processing of image sequences was done using the ImageJ/FIJI software.