Cells were replated on Matrigel-coated 8-well μ-slides (Ibidi) using EDTA. After 48 h, calcium transients were measured using the Fluo-4 Direct Calcium Assay kit (Thermo Fisher Scientific). Fluo-4 was prewarmed at 37°C and loaded into the cells by adding an equal volume to the culture medium present in the well. Cells were incubated at 37°C for 30 min. In experiments, using chemical receptor agonists, isoproterenol hydrochloride (Sigma) or carbamoylcholine chloride (Sigma) were diluted in prewarmed Fluo-4 solution and loaded into cells at a final concentration of 1 μM. Cells were incubated at 37°C for 15 to 30 min.
Samples were measured using the confocal microscope previously described with a dry objective 10.0 × magnification (numerical aperture of 0.40), with images obtained at a resolution of 128 × 128 pixels, with a scan speed of 700 Hz, and with frame intervals of 97 msec during 60-120 sec. Fluo-4 excitation was performed using a 488 nm line with an Argon ion laser and fluorescence emission was collected (500-650 nm) using a Leica HyD hybrid detector. Samples were measured within 4-5 min after leaving the incubator using a heating stage set at 37°C to maintain the temperature before and during measurements.
Samples were measured using the confocal microscope previously described with a dry objective 10.0 × magnification (numerical aperture of 0.40), with images obtained at a resolution of 128 × 128 pixels, with a scan speed of 700 Hz, and with frame intervals of 97 msec during 60-120 sec. Fluo-4 excitation was performed using a 488 nm line with an Argon ion laser and fluorescence emission was collected (500-650 nm) using a Leica HyD hybrid detector. Samples were measured within 4-5 min after leaving the incubator using a heating stage set at 37°C to maintain the temperature before and during measurements.