Isolation of Adult Cardiac Cells

Adult hearts were transcardially perfused with PBS containing 0.9 mM Ca²⁺, and atria and valves were removed. Single-cell suspensions were obtained as previously described (Pinto et al., 2016 (link)). Briefly, tissue was minced and incubated with collagenase type IV (600 U/mL, Worthington, LS004188) and dispase II (1.2 U/mL, Thermo Fisher, 17105041) in DPBS containing 0.9 mM Ca²⁺ for 45 min at 37°C and filtered through both a 40 μm and then a 30 μm filter to ensure a single-cell suspension. Yellow amine-reactive live/dead cell dye (Thermo Fisher, L34959) or LIVE/DEAD Fixable Violet Dead Cell Stain kit (Thermo Fisher, L34964) was used for live/dead discrimination. Cells were then incubated in Fc block (1:100, Tonbo Bioscience, 70–0161 U500) followed by indicated antibodies and fixed with 1% PFA in flow cytometry buffer. Primary antibodies used for flow cytometry are listed in Supplementary file 1f. Accucheck counting beads (Thermo Fisher, PCB-100) were added according to the manufacturer’s protocol prior to acquisition. Data were acquired on an LSRFortessa (BD Bioscience) and analyzed using FlowJo software version 10 (BD Bioscience). Cell counts were calculated according to the Accucheck counting bead manufacturer’s recommendations and normalized to the weight of individual ventricles.