Calcium Imaging and Cell Counting Protocol

Ca²⁺ imaging was performed following standard protocols [8 (link)]. Briefly, cells were labeled with Rhod-3 from a Calcium Imaging Kit (Thermo Fisher Scientific, R10145) for 1 h at room temperature, washed, and incubated for an additional hour, to allow de-esterification of the dye. Rhod-3-labeled cells were analyzed at 37 °C using an LSM 510 META confocal microscope (Carl Zeiss, Oberkochen, Germany). Ca²⁺ oscillation⁺ cells were counted in ten randomly selected fields per well in at least three independent experiments. To count the number of beating cells, we seeded 50,000 fibroblasts per well on 12-well plates, performed cell transductions, cultured the cells with the indicated media, and then monitored cell contraction. For accurate analyses of the cell count, we used an all-in-one fluorescence microscope as described previously (BZ-9000; Keyence, Tokyo, Japan) [8 (link)]. We first acquired images of the cells in all the areas in a well with a 20× phase contrast lens by moving the motorized stage sequentially. We next moved the field to cover all the areas in a well, and counted the number of spontaneously contracting cells in each field with the 20× phase contrast lens in at least three independent experiments. The measurements and calculations were conducted in a blinded manner.