Directed migration of dendritic cells toward a CCL19 gradient in a planar under-agarose assay. (A) Analysis of DC chemotaxis in a planar under-agarose assay. DCs migrate beneath the agarose toward the 1-mm afar attractor hole containing 1.2 μg/mL CCL19. Directed migration at 37°C, 5% CO₂ was recorded by time-lapse videomicroscopy and analyzed with ImageJ software (in panels B-E). (B) Tracks of single DCs migrating toward a CCL19 gradient over 3 hours (n = 30 each). WT indicates wild-type. (C) Comparison of velocity (line: median, U test, P < .001) and (D) directionality (line: mean, t test, P < .001) of chemotaxing DCs. Cells were derived from 3 independent BM DC cultures (of both WT and Cdc42^−/− mice) and applied to individual experiments. Forty single cells (dots) per experiment were tracked (WT: n = 120, Cdc42^−/−: n = 120). ***P < .001. (E) Correlation of morphology and migration tracks of representative single WT and Cdc42^−/− DCs migrating along a CCL19 gradient. The top track shows outlines of DC morphology over time (WT: 82 minutes, Cdc42^−/−: 177 minutes). Morphology outlines of DCs were obtained by morphometric analysis (MetaMorph). The middle track (black line only) represents the migration track, whereas the bottom track indicates migration phases with instantaneous velocities over a threshold speed of 7 μm/minutes (gray shaded). Scale bar represents 50 μm. Objective: A-Plan 10×/0.25 Ph1 (Zeiss).