Percp cy5.5 cd86

One day after IR, hearts were perfused with pre‐cold PBS. Left ventricle tissues subjected to IR injury were removed and dissociated with gentleMACS dissociator (Miltenyi Biotec, USA). The digestion was performed in 5 ml HBSS buffer contained Collagenase II (Worthington, 1.5 mg/ml), Collagenase IV (Worthington, 1.5 mg/ml) and DNase I (Sigma, 60U/ml) at 37℃ for 30 min at a speed of 200 rpm. The resulting suspension was filtered (70 μm) to generate a single‐cell suspension. The suspension was centrifuged at 300 × g for 5 min. Cardiac cells were then resuspended in DMEM media (supplemented with 10% FBS and 1% Penicillin‐Streptomycin) and cultured for 2 h (37°C, 5% CO2). Cells were then washed with PBS and cardiac macrophages were enriched in the adherent cells (de Couto et al., 2017). Cells were collected and incubated with flow cytometry antibodies, including FITC‐CD4, PE‐F4/80, PerCP‐Cy5.5‐CD86 and PE‐Cy7‐CD206 (all from BioLegend, USA) at 4°C in the dark for 15 min. After PBS washing, the phenotype of cardiac macrophages was detected by BD FACSCanto II flow cytometer (BD Bioscience, USA).