All conventional DC subsets respond to curdlan in vivo. (A) Expression of dectin-1 on spleen CD11c⁺CD8α⁻ and CD11c⁺CD8α⁺ cells. Splenocytes were stained with anti–dectin-1 (thick line) or rat IgG2b (solid gray). DC subtypes were defined by gating as shown on the dot plot (left panel). Numbers indicate percentages of events in gate (dot plot) or mean fluorescence intensity (GeoMFI; histograms). (B) Expression of dectin-1 on lymph node DC subsets. Cell suspensions from peripheral lymph nodes were stained as in panel A. Blood-derived resident CD11c⁺CD40^loCD8α⁻ or CD11c⁺CD40^loCD8α⁺ cells and skin-derived migratory CD11c⁺CD40^hi subsets were defined by gating as shown on the dot plots (upper panels). (C) BMDCs were incubated with fluorescein-curdlan and analyzed by confocal microscopy. Note the fluorescein signal within the cells. (D) Mice were injected in both hind footpads with 1 mg fluorescein-curdlan or PBS 6 hours before popliteal lymph nodes were removed and analyzed by flow cytometry. Dot plots show fluorescein signal on resident DCs (CD11c⁺CD40^lo, left) and migratory DCs (CD11c⁺CD40^hi, right) plotted against expression of CD8α. (E) Mice were injected in both hind footpads with 200 μg curdlan, 10 μg polyI:C or PBS 6 hours before popliteal lymph nodes were removed and analyzed. Data are GeoMFI of dectin-1 (top) and CD86 staining (bottom) on skin-draining DCs (CD11c⁺CD40^hi, left), and CD8α⁻ and CD8α⁺ lymph-node resident DC subsets (CD11c⁺CD40^lo, middle and right).