Figure 1.

Gating strategy for identifying the different immune populations in the heart. Mononuclear cells expressing CD45 were gated and doublets (FSC-W vs. FSC-A) were excluded. Dead cells were excluded by Zombie aqua. The live single CD45⁺ cells were then grouped into R1, CD11b+ myeloid cells (CD45⁺/CD11b⁺/CD11c⁻); R2, dendritic cells (CD45⁺/CD11b⁺/CD11c⁺); and R3, NK cells (CD45⁺/CD11b⁻/CD11c⁻/NK1.1⁺) based on their relative expression of CD11b and CD11c. R5, neutrophils (CD45⁺/CD11b⁺/CD11c^-/Ly6G^hi) were then excluded from R1 based on their Ly6G expression. The remaining R4 monocytic cells were then further characterized into R6, Ly6C^hi or commonly known as M1 cells (CD45⁺/CD11b⁺/CD11c⁻/Ly6G^lo/Ly6C^hi); R7, Ly6C^lo or commonly known as M2 cells (CD45⁺/CD11b⁺/CD11c⁻/Ly6G^lo/Ly6C^lo) based on their Ly6C expression; and into R8, fetal liver HSC-derived resident macrophages (CD45⁺/CD11b⁺/CD11c^-/Ly6G^lo/CCR2⁻/MHC-II^hi); R9, monocyte derived macrophages (CD45⁺/CD11b⁺/CD11c^-/Ly6G^lo/CCR2⁺/MHC-II^hi); R10, monocytes (CD45⁺/CD11b⁺/CD11c⁻/Ly6G^lo/CCR2⁺/MHC-II^lo); and R11, yolk sac-derived resident macrophages (CD45⁺/CD11b⁺/CD11c⁻/Ly6G^lo/CCR2⁻/MHC-II^lo) based on their CCR2 and MHC-II expression. These CCR2 and MHC-II gated populations were then back gated on R6 and R7 and their relative contribution to the M1 (Ly6C^hi) and M2 (Ly6C^lo) cells was assessed.