Analysis of ex vivo dendritic cell migration in a dermal ear explant. (A) Ears of C57BL/6 mice were separated into halves and mature DCs placed on top of the dermis. Two hours after addition of TAMRA-labeled DCs (red dots) and incubation at 37°C, 5% CO₂, entry of DCs into deep lymphatics was analyzed (in panels B-D). (B top row) Wild-type and (bottom row) Cdc42^−/− DCs were placed on dermal ear explants in separate experiments. One percent PFA-fixed ear sheets were counterstained with anti–LYVE-1 antibody (green) to detect lymphatic vessels in the dermis. The images represent merged z-stacks (WT: 18 planes, 1.1 μm z-steps; Cdc42^−/−: 24 planes, 1.3 μm z-steps) obtained by confocal microscopy (Leica DMIRE2). Scale bars represent 100 μm. Objective: HCX PL APO/40×/1.25-0.75 oil (Leica). (C) Quantification of immunofluorescence analysis. Cells were derived from 2 independent BM DC cultures (of both WT and Cdc42^−/− mice). Single DC cultures were applied to 3 C57BL/6 ears, confocal z-stacks 3-dimensional (3D) reconstructed, and the number of DCs within lymphatics calculated by morphometric analysis (mean ± SD; t test, P < .001, WT: n = 6, Cdc42^−/−: n = 6). ***P < .001. (D) Side view of the ear dermis (z = 50 μm) after addition of a 1:1 mixture of WT (red) and Cdc42^−/− (green) DCs.