Ixon 860

Electrically-stimulated Ca²⁺ transients were recorded from engineered muscle bundles after 1, 2, 4 week of in vitro culture and from muscle explants 7–15 days post implantation, as previously described^{6 (link),37 (link)}. In vitro cultured iSKM bundles and excised dorsal skins and TA muscles with engrafted bundle implants were transferred into a custom chamber mounted on an upright fluorescence microscope (Leica M165 FC, for TA muscle explants) or an inverted fluorescence microscope (Nikon TE2000-U, for window chamber explants), placed in 37 °C differentiation media (DM, Supplementary Table 1), and electrically stimulated (10 ms pulse, 3 V/mm). Resulting GCaMP6 (510–560 nm bandpass emission filter) or R-GECO (590-660 nm bandpass emission filter) signals were recorded using a fast EMCCD camera (Andor iXon 860; 24 µm spatial and 20 ms temporal resolution). Amplitudes of Ca²⁺ transients were determined using the Solis software (Andor) by averaging relative fluorescence intensity (ΔF/F) from each bundle.