Supplementary Figure 1: Phenotypic characterization of colonic macrophages and Kupffer cells in neonatal and adult mice.

(a) Absolute numbers of total live CD45⁺ cells obtained from enzymatic digests of colon from newborn (day 2), day 7, day 14, day 21 and 7-8 week old adult CX3CR1^+/gfp mice. Data were obtained from 3-6 individual mice per group. (b) Gating strategy used to identify F4/80⁺ CD11b⁺ cells amongst colon digests. Cell aggregates and dead cells were excluded on the basis of FSC and 7-AAD staining respectively. Leukocytes were then selected by their expression of CD45, before granulocytes were excluded by gating out SiglecF⁺ eosinophils and Ly6G⁺ neutrophils. Mucosal DC were excluded on the basis of their high levels of CD11c expression and lack of F4/80. (c) Expression of CD11c by F4/80⁺ CD11b⁺ colonic LP cells from 2 day old mice (black line) and adult mice (green line), and by total CD45⁺ leukocytes from adult colon (red line). (d) Quantitation by qRT-PCR of mRNA for CD163 by FACS-purified F4/80^hi LP cells from resting colon of newborn (day 0-1) or adult mice, and CSF-1 generated BM-derived macrophages (BMM). Results are shown as mean expression relative to cyclophilin A (CPA), using the 2^-ΔdC(t), with BMM set to 1. (e) Expression of MerTK by F4/80⁺ CD11b⁺ cells from colon of 2 day old mice and adult mice, and by F4/80^hi CD11b^lo liver Kupffer cells. (f) Expression of CX3CR1-GFP and CD64 by F4/80^hi CD11b^lo (red line) and F4/80^lo CD11b⁺ (blue line) cells from colon of 2 day old CX3CR1-GFP reporter mice. (g) Gating strategy used to identify total F4/80⁺ CD11b⁺ cells amongst liver digests. Cell aggregates and dead cells were excluded on the basis of FSC and 7-AAD staining. CD45⁺ leukocytes were selected and granulocytes excluded by gating out SiglecF⁺ eosinophils and Ly6G⁺ neutrophils. Liver DC were excluded on the basis of their high levels of CD11c and MHCII expression.