Multiparametric Flow Cytometry of Cardiac Immune Cells

Peripheral blood (50 μL) was taken immediately after cervical dislocation for flow cytometric analysis. RBCs were lysed with 2 mL ACK lysing buffer (Gibco, Thermo Fisher Scientific, A10492-01) for 10 minutes. Excised LV infarcted tissue was rinsed and immediately minced and digested by warm digestion buffer containing collagenase type 2 (0.05%, Worthington Biochemical, LS004177) and DNase 1 (60 U/mL DNase 1, MilliporeSigma, 10104159001), followed by shaking for 5 minutes in 37°C. A single-cell suspension was generated and filtered through a 40 μm filter into ice-cold stopping buffer containing 10% FBS in 5 mL PBS. This process was repeated, with the tissue remaining in the filter for a total of 5 digestions. Cells were centrifuged (400g, 5 min), and RBCs were lysed with 2 mL ACK lysing buffer (Gibco, Thermo Fisher Scientific, A10492-01) for 5 minutes. Cells were then washed twice with PBS. Immune cells isolated from blood and infarcted heart tissue were resuspended in 100 μL staining buffer (1% PBS in 2 mM EDTA). The cells were incubated with 1% CD16/CD32 for 10 minutes at room temperature followed by incubation of a 1% antibody mixture on ice for 20 minutes. Cells were then washed twice with iced PBS, counted, and fixed. For flow cytometric analysis, data were acquired with an Aurora flow cytometer (Cytek) and analyzed with FlowJo 10.7.1 software (FlowJo). Viability stain 7-AAD was used to identify live cells. The gating strategies are shown in Supplemental Figure 6 (neutrophils: CD45⁺CD11b⁺ly6G⁺, monocytes: CD45⁺CD11b⁺ly6G^–, ly6C^hi monocytes: CD45⁺CD11b⁺ly6G^–ly6C^hi, and CD206⁺ macrophages: CD45⁺CD11b⁺ly6G^–CD206⁺). Antibodies against CD45-BV421, CD11b-APC, Ly6G-APCcy7, Ly6C-AF700, and CD206-PE were used for flow cytometry (see Supplemental Table 2 for details). For cM cell separation, an immunomagnetic positive selection cell isolation kit was used (catalog 18970, EasySep).