Immature lymphoid organ DCs acquire a mature phenotype during culture in vitro. (A) Left: FACS analysis of CD86 and MHC II expression in splenic DCs freshly isolated (0 time point, thinnest continuous line) or after incubation for the times indicated. The results were similar for each individual splenic DC population. The FACS profiles of MHC II expression at 6 and 18 hours were comparable, so the latter has been removed for clarity. The dashed line corresponds to unlabeled cells. The result shown is representative of multiple experiments. Right: FACS analysis of the expression of MHC II and CD86 in BMDCs after incubation without (thin line) or with (thick line) LPS for 20 hours. The dashed line corresponds to unlabeled cells. The result shown is representative of multiple experiments. (B) Immunofluorescence confocal microscopy (ICM) analysis of MHC II (green) and Lamp 1 (red) localization in BMDCs incubated for 20 hours in the absence (immature, left panels) or the presence (right) of 1 μg/mL LPS. (C) Subcutaneous LN epDCs and splenic CD8⁺ DCs were purified and analyzed by ICM immediately after isolation (“Fresh”) or after culture for 18 hours in vitro (“Cultured o/n”). Cells were stained with antibodies for MHC II (I-A^b, red), the late endosomal/lysosomal marker Lamp1 (green), and the nuclear dye DAPI (blue).