Figure 1: CD169⁺ macrophages in the lamina propria.

(a) Flow cytometry of LP myeloid cells in the colon. CD11b⁺ and/or CD11c⁺ (‘myeloid’) cells were enriched by magnetic sorting and stained for Ly6C, CD64, CD169 and Siglec-F or CD103. Dead cells were excluded by 7AAD staining. Numbers indicate frequencies of CD64^lo, CD64^int, CD64^hi, Siglec-F⁺ and CD103⁺ fractions among 7AAD⁻ cells (left). Frequencies of CD169⁺ cells in each fraction are presented (right). Dashed lines represent isotype control. (b) Flow cytometry of LP myeloid cells in the colon of CX3CR1^gfp mice. Numbers indicate frequencies of CX3CR1^neg, CX3CR1^int and CX3CR1^hi fractions among 7AAD⁻ myeloid cells (left). Frequencies of CD169⁺ cells in CX3CR1^neg, CX3CR1^int and CX3CR1^hi fractions are presented (right). Dashed lines represent unstained control. (a,b) Representative of three independent experiments. (c) Subfractionation of LP myeloid cells in the colon according to the expression of CD11b and CD11c. Fifteen percent of the 7AAD⁻ myeloid cells expressed CD169 (top right). The specificity of anti-CD169 antibody was confirmed by staining LP myeloid cells of CD169 knockout (KO) mouse (bottom left). LP myeloid cells were subdivided into four subpopulations (R1–R4) based on the differential expression of CD169, CD11b and CD11c. Numbers indicate frequencies among 7AAD⁻ myeloid cells. Representative of five independent experiments. (d) Flow cytometry analysis of a panel of surface molecules on CD169⁺ and CD169⁻ cells in the colon. Shadow represents unstained control. Representative of two independent experiments. (e) CD169⁺ macrophages were localized distantly from the epithelial border. Consecutive colon sections from CX3CR1^gfp mice were stained for GFP (left) or CD169 (middle). Original magnification, × 20. Scale bars represent 100 μm. The distance of CX3CR1⁺ cells or CD169⁺ cells from the epithelial border was quantified (right). *P<0.05, Student’s t-test. Representative of two independent experiments. DAPI, 4,6-diamidino-2-phenylindole.