Dermal DCs are significantly reduced in the absence of GM-CSF and FL in steady state. DCs were analyzed from WT, GM-CSF^−/−, FL^−/−, and DKO mice. (A) Immunofluorescence microscopy of MHCII-stained (red) epidermal sheets. MHCII⁺ cells were counted on images taken from multiple fields per mouse (n = 4/group). Epidermal sheets were stained with PE-conjugated anti-MHCII antibody and mounted on slides with the use of Eukitt mounting medium. Images were taken at room temperature on a Nikon Eclipse E800 microscope with a CCD Qimaging camera using a Nikon Plan Apo 20×/0.75 NA objective lens and acquired with the use of OpenLab software. Scale bar represents 10 μm. (B-C) Flow cytometry of ex vivo–isolated dermal-derived cells. Representative FACS plots from WT, GM-CSF^−/−, FL^−/−, and DKO mice are shown. (B) Percentage of all CD45⁺ cells shown by outer gate. Inner gate represents percentage of MHCII⁺ cells within CD45⁺ gate. (C) Dermal DCs were pregated on MHCII⁺CD45⁺, followed by gating on CD11b⁺ and CD11b⁻ populations. (D) Percentages of total dermal-derived MHCII⁺CD45⁺ cells, as well as CD11b⁻ and CD11b⁺ subsets are shown. Results are given as the percentage of CD45⁺ cells (n = 4-6 mice/group). *P < .05; **P < .01.