Confocal Calcium Imaging of hiPSC-Cardiomyocytes

Confocal Ca²⁺ imaging experiments were performed with an Olympus Fluoview laser-scanning confocal microscope (Olympus Life Science, Center Valley, PA, USA) as previously described [18 (link),41 (link)]. Fluo 4-AM [dissolved in 20% F-127 pluronic in dimethyl sulfoxide (DMSO), final concentration 15 µM] was added to hiPSC-CMs and incubated for 20 min at room temperature. Fluo-4 loaded hiPSC-CMs were placed in a perfusion chamber and excited at 488 nm using an argon laser, and fluorescence emission was detected via a 520-nm band-pass filter and photomultiplier tube. Confocal images were acquired with the Fluoview acquisition software program and spontaneous activity was recorded on a personal computer for later analysis. Images acquired with Fluoview acquisition software were analyzed with ImageJ and Fluoview analysis software (Olympus Life Science, Center Valley, PA, USA).