Measuring Intracellular Calcium Dynamics

NRCMs were plated on coverslips in 3-cm dishes for routine cultivation. After carrying out all intervention protocols, NRCMs were eluted with Hanks Balanced Salt Solution (HBSS), and then incubated with 5 μM/mL Fura 2-AM (Dojindo Laboratories, Kumamoto Japan) in DMSO and diluted in HBSS for 30 min at 37 °C followed by another HBSS elution. NRCMs on coverslips were then sent for calcium transient recording using a dual-excitation fluorescence photomultiplier system (IonOptix, Milton, MA, USA). Briefly, the NRCMs were continuously covered with warm oxygenated Tyrode's solution (140 mM NaCl, 1 mM MgCl₂, 6 mM KCl, 10 mM glucose, 2 mM CaCl₂, and 5 mM HEPES, pH 7.4) [35 (link)]. Fluorescence intensity at 510 nm was detected with alternate scanning by fluorescence emissions at 340 nm and 380 nm. Intracellular Ca²⁺ levels and the intracellular Ca²⁺ transient decay time constant were recorded and analyzed by the SoftEdge MyoCam® system (IonOptix).