Cardiac Leukocyte Characterization by Flow Cytometry

Total cardiac cells were gated on PerCP-conjugated CD45 (BD Biosciences), and the following antibodies were used: PE-Cy7–conjugated anti-CD11b (BD Pharmingen), PE-conjugated anti-Ly6G (lymphocyte antigen 6 complex, locus G; 1A8; BD Pharmingen), FITC-conjugated anti-Ly6C (lymphocyte antigen 6 complex, locus C; Biolegend), PE-Alexa700–conjugated anti-CD3 (eBioscience) and APC-conjugated anti-F4/80 (Bio-Rad). CD45⁺/CD11b⁺ leukocytes were gated on F4/80⁻ monocytes and then further stratified by Ly6G and Ly6C expression. Monocytes were identified as CD11b^highLy6G⁻Ly6C⁺. Neutrophils were identified as CD11b⁺Ly6G^high and T cells as CD45⁺CD3⁺. The total number of cells was then normalized to LV weight. Cells were analyzed using a flow cytometer (LSR II; BD Biosciences).^{10 (link),11 (link)}