Transmigration of a single human monocyte on activated HUVECs under flow. (A) Human monocytes that adhere out of free flow typically activate within 1 minute, changing from a phase-white (0 minutes) to a phase-gray appearance (2 minutes) before migrating on luminal surfaces (5 minutes) to an endothelial cell junction (white arrows). Transmigration between the lumen and ablumen (7 minutes) leads to a change in phase appearance (black) as the monocyte migrates under the HUVEC monolayer (11-12 minutes). (B) There was a reduction in adherent monocytes occupying the ablumen on HUVECs with blocking mAbs to JAM-C. A 5-minute bolus of monocytes was flowed over activated HUVECs at a shear stress of 0.05 Pa. Adherent monocytes were cocultured under continued flow with the HUVECs for 20 minutes before monocytes occupying the lumen (phase-gray) and ablumen (phase-black) were counted. Abluminal monocyte counts were presented as a percentage of total adherent monocyte per field. Each group is expressed as the mean of triplicate cell counts (± SEM; **P < .01).