Cardiac Immune Cell Profiling

At 3 h post-study, a subset of animals (n = 6 naïve, n = 5 sham-subjected to vessel cannulation and terminal anesthesia only, n = 5 THS) were intracardially perfused with heparinized saline under terminal anesthesia. The hearts were then immediately isolated, cut into 1 mm³ pieces and dissociated by incubation with papain (Merck, UK) and DNAse I (ThermoFisher Scientific, UK) for 30 min at 37°C. Following lysis of residual red blood cells (RBC Lysis Buffer, Biolegend, UK), cell suspensions were incubated with CD16/CD32-block (Biolegend, UK) for 30 min at 4°C, followed by incubation for 30 min at 4°C with PE-conjugated anti-CD45 to define immune cell populations, and FITC-conjugated anti-Ly6C/G (clone RB6-8C5) and APC-conjugated anti-F4/80 (all ThermoFisher Scientific, UK) to differentiate neutrophils (F4/80Neg, Ly6C/GHi) from pro-inflammatory (F4/80Pos, Ly6C/GInt) and anti-inflammatory (F4/80Pos, Ly6C/GLow) monocytes/macrophages (19 (link), 20 (link)). Cells were then analyzed by flow cytometry using a BD FACSCanto II instrument (BD Biosciences) and FlowJo 8.8.1 software (TreeStar Inc., FL, USA). In all cases, 20,000 singlet CD45Pos events were analyzed per sample; positive staining was defined by inclusion of fluorescence-minus-one controls for all antigens.