Gating strategy for immune cells in the mouse lung. (A) Gating strategy to identify lung myeloid cells in Gpr183^GFP/+ reporter mice (see Fig. 1 A and Fig. 2, A and B). Lung cells were first gated on live single CD45⁺ lineage (CD3/CD19/NK1.1)⁻ cells before separating myeloid cells into the indicated CD64⁺ macrophage and CD64⁻ cell populations as shown. Alveolar macrophages in BAL fluid (Fig. 2, A and F) were gated as CD64⁺Ly6G⁻CD11c⁺SiglecF⁺ cells as shown here for alveolar macrophages in the lung. AMs, alveolar macrophages; Eosino, eosinophils; FSC-A, forward scatter area; FSC-H, forward scatter height; FSC-W, forward scatter width; IMs, interstitial macrophages; PMNs, polymorphonuclear neutrophils; SSC-A, side scatter area. Data are representative of at least three independent experiments with a total of n = 5–6 mice. (B) Gating strategy to identify lung macrophage populations in chimeras 3 wk after bone marrow reconstitution (see Fig. 2 E and Fig. 7 A). Cells were pre-gated as live single CD45⁺lineage⁻CD64⁺Ly6G⁻ cells as in panel A and then separated into the different macrophage populations. Mono-mac, monocyte-macrophages. Data are representative of at least two independent experiments with a total of n = 4–13 mice. (C) Gating strategy to identify lung immune cells in the steady-state lung of Gpr183^+/+ and Gpr183^−/− mice (Fig. 2 C) and in the lung of bone marrow chimeras (Fig. 4, A–D; Fig. 6, B and D; and Fig. 7 C). After pre-gating on live single CD45⁺lineage⁻CD64⁻, CD45⁺lineage⁻CD64⁺, or CD45⁺lineage⁻ cells as in panel A, the indicated cell populations were gated as shown. Data are representative of at least two independent experiments with a total of n = 4–19 mice.