DOCK8 is required for DC migration in 3D, but not 2D, environments. (A) Localization of Dock8^+/− and Dock8^−/− DCs within the dermis of ear explants after a 90-minute incubation. Scale bar indicates 100 μm. (B) Comparison of the velocity of Dock8^+/− and Dock8^−/− DCs migrating within the dermis of ear explants. Velocities were determined by analyzing more than 160 cells per group. **P < .01. (C) Migration of LPS-stimulated Dock8^+/− (n = 232) and Dock8^−/− (n = 194) DCs toward 60 μL of CCL21 source (10 μg/mL) in 3D collagen gels. DC migration was recorded for 120 minutes by time-lapse videomicroscopy. **P < .01. (D) EZ-Taxiscan chemotaxis assay of LPS-stimulated Dock8^+/− (n = 47) and Dock8^−/− DCs (n = 53) toward 1 μL of CCL21 source (250 μg/mL) on a fibronectin-coated 2D surface. DC migration was recorded for 30 minutes by time-lapse videomicroscopy. (E) Morphology of Dock8^+/− and Dock8^−/− DCs migrating toward a CCL21 gradient in 3D collagen gels. SYTO12 was used to stain nuclei (green). Elapsed time is presented as minutes:seconds. Scale bar indicates 10 μm. In panels B through D, each box plot exhibits the median (central line within each box), the 25th and 75th percentile values (box ends), and the 10th and 90th percentile values (error bars). Data are representative of 3 (A,C,E) or 2 (D) independent experiments and are from 3 (B) separate experiments.