Phenotypic characterization of colonic mononuclear phagocytes. (a) Doublets were excluded from colonic digests from CX3CR1gfp/+ mice on the basis of FSC-A and FSC-H, and live leukocytes were selected as being 7-AAD− CD45+. The resulting cells were then analyzed for CD11b and CX3CR1-green fluorescent protein (GFP) expression. (b) Histograms show the expression of the indicated cell surface markers on the CX3CR1−, CX3CR1int, and CX3CR1hi subsets. (c) Expression of Ly6C, MHCII, F4/80, and CD11c on CX3CR1int (top panels) and CX3CR1hi CD11b+ cells (bottom panels). (d) Morphological assessment of fluorescence-activated cell-sorted P1, P3, and P4 cells (× 200). (e) Phagocytic activity of colonic MP cell subsets, as measured by the uptake of pHrodo E. coli bioparticles for 15 min at 37 °C (black lines) or at 4 °C as a control (shaded histograms). Representative pHrodo dye fluorescence of CX3CR1-based P1 to P5 and CD103+ DC. Mean ΔMFI (mean fluorescence intensity (MFI) of pHrodo dye fluorescence at 37 °C-MFI at 4 °C control) for populations 1 to 4 + 1 s.d. for 4–5 mice is shown. Representation of three individual experiments (***P<0.001).