Isolation of Cardiac Immune Cells

Single-cell suspensions were prepared as described previously (12 (link), 28 (link)). Briefly, mice were deeply anesthetized and intracardially perfused with 20 ml of ice-cold PBS to eliminate blood cells. The hearts were minced with fine scissors and placed into a cocktail of 1 mg/ml collagenase II (Worthington, Lakewood, NJ, USA), 100 U/ml elastase (Worthington), and 100 U/ml DNase I (Sigma-Aldrich) and shaken at 37°C for 1 h. Tissue samples were then triturated through a 70 μm cell strainer and centrifuged (5 min, 400 g, 4°C). The obtained cells were counted after erythrocyte lysis and washed using PBS for further analysis. For staining, 5 × 10⁶ cells were pre-incubated for 5 min on ice with anti-CD16/CD32 antibody (2.4G2, BD Bioscience, San Jose, CA, USA) to block the non-specific antibody and then stained with directly conjugated antibodies for 30 min at 4°C in the dark in PBS. For intracellular cytokine staining, single-cell suspensions were stimulated with 50 ng/ml (PMA, Sigma-Aldrich), 1 μg/ml ionomycin (Sigma-Aldrich), and Golgi-PlugTM (BD Biosciences) for 4 h. Surface staining was performed first. After fixation and permeabilization using the Cytofix/Cytoperm Soln kit (BD Biosciences), intercellular proteins were stained. All experiments were performed on an Attune NxT flow cytometer (Invitrogen) and analyzed using FlowJo version 10 software.