Schematic presentation of modified transwell chamber assay system for monitoring lymphocyte TEM under flow. (A, left) Classical Boyden assay setup (Trans-well Chamber) wherein lymphocyte chemotaxis across endothelial barrier (or filter) toward a chemoattractant in the lower chamber is quantified (lymphocyte readout). (A, right) Modified transwell chamber wherein lymphocytes are perfused over an endothelial monolayer with or without apical chemokines, with additional chemoattractant(s) introduced beneath the endothelium (basal presentation). TEM is measured in real time by video microscopy under fluid shear conditions (TEM is quantified as lymphocytes collecting underneath the endothelium). (B) T-cell chemotaxis across TNF-α–activated HUVECs toward 100 ng/mL CCL5 introduced in the lower chamber under static conditions. Apical CXCL12 (10 ng/mL) was overlaid for 5 minutes and then was washed before lymphocyte placement. Data are mean ± SEM from 3 independent experiments. *P < .05.