Defective DC mobilization from skin to draining LNs in ICSBP^-/- mice upon FITC sensitization in vivo. (A-B) Mice were sensitized with FITC on the shaved abdomen. Twenty-four hours later (A), draining inguinal LNs were removed and cell suspensions were subjected to Nycodenz centrifugation to enrich for DCs. Cells were then stained with PE-labeled anti-CD11c antibody and analyzed by FACS for the presence of DCs that had migrated from the skin (FITC⁺). Density plots were gated to exclude dead cells. (B) Draining LNs were removed 24 hours or 48 hours after skin sensitization with FITC. Histograms represent the expression of FITC (M1) on cells gated on forward-side scatter properties (R1) and on CDIIc positivity (R2). (C) Draining lymph nodes from 48-hour FITC-treated mice were enriched on Nycodenz and double-stained with anti-CD11c coupled with anti-CD40 or anti–I-A, alternatively. Dot plots and histograms show the population gated on the basis of FITC positivity. (D) FITC was applied to ears of ICSBP^-/- and WT mice. After 24 hours, epidermal sheets were incubated for an additional 24 hours in medium containing GM-CSF to allow the release of epidermal cells from the tissue. Cells emigrated in the culture medium were collected, counted, and subjected to FACS analysis. Histograms represent the number of cells expressing the indicated markers in cultured epidermal sheets of untreated (□) or FITC-treated (▦) mice. Representative data of 1 of 3 separate experiments are shown.