M 1 thioglycerol

E 15.5, P2, P4, and adult cardiac cells were isolated as above, and HSA⁺ cells were sorted following the gating strategy in S1A Fig. For the neonatal and adult cells, 20 mM BDM (Sigma-Aldrich) was added throughout the isolation procedure [48 (link)]. HSA⁺ cells were plated in 0.1% collagen (Life Technologies) for E 15.5 or in fibronectin/gelatin coated ibidi plates for postnatal cells, and cultured for 1 week in high-glucose Iscove's Modified Dulbecco's media (Life Technologies) supplemented with 20% FBS, 1× penicillin/streptomycin (Life Technologies, USA), 1× L-glutamine (Life Technologies), 50 μg/mL ascorbic acid, and 1.5 × 10⁻⁴ M 1-thioglycerol (Sigma-Aldrich), as previously described by Wu and colleagues [4 (link)]. Adult cardiac cells were incubated at 37°C in 3% O₂. Live-cell imaging was performed on a temperature-controlled Zeiss Axiovert 200M microscope equipped with a CoolSnap HQ (Roper) camera (Zeiss, Germany). Sample position was controlled by an X-Y motorized stage, and images were acquired every 15 minutes using an A-Plan 20×/0.30 objective for 48 hours.