Rac GTPases are required for chemokine-induced adhesion to, migration on and transmigration through endothelium. WT and DKO T cells were allowed to attach to an endothelial monolayer at 0.24 dyne/cm² for 5 minutes, and flow was then increased to 1 dyne/cm² (high shear stress). Behavior of EYFP⁺ T cells was recorded by video microscopy. (A) Graph shows mean ± SEM percentage of EYFP⁺ T cells remaining attached on top of the endothelium after 5 minutes of high shear stress (either crawling or stationary) as a fraction of EYFP⁺ T cells at the start of the high shear stress period. (B) Flower plots show migration tracks of individual T cells of the indicated genotype, either on top of or below the endothelium, with the starting point of each track placed at the origin (0, 0). Cells that left the field of view during recording were not included. Note that in DKO plots there 44 and 39 cell tracks shown on top and below endothelium, respectively, but most are not clearly visible as they remain close to the origin. (C) Scatter plot of distance traveled by T cells on top of the endothelium. (D) Scatter plot of mean velocities of individual T cells on top of the endothelium. (E) Scatter plot of distance traveled by T cells below the endothelium. (F) Scatter plot of mean velocities of T cells below the endothelium. (G) Graph showing mean ± SEM percentage of T cells that had transmigrated through the endothelium by the end of the video recording (up to 55 minutes), as a fraction of EYFP⁺ T cells at the start of the high shear stress period. In panels C-F, red lines indicate means. Data are from 5 videos analyzed from 3 independent experiments. *P < .01; ***P < .001.