Localization of PMNs on the apical surface of HUVECs. PMNs from healthy donors were labeled with CFSE, incubated with anti-CD157 or anti-HLA class I mAb, and seeded on TNF-α–activated HUVEC monolayers grown on collagen. After 1 hour, samples were stained with silver nitrate to identify endothelial cell junctions. These representative apical views show where neutrophils are found, but are not meant to represent the results of the assays quantitatively. In the presence of the isotype-matched control mAb (A) neutrophils are below the endothelial monolayer (white arrows) and few cells are located on the apical surface of HUVECs. CD157 ligation leaves most of neutrophils on the apical surface (B), mainly accumulated at endothelial cell junctions and at tricellular corners. A limited number of cells crossed the endothelial layer (white arrows). Representative images are shown. Samples were observed simultaneously by DIC and fluorescence confocal microscopy. Cells were imaged using a × 60 oil immersion objective (1.4 NA; bar represents 30 μm). The routes of several transmigrating neutrophils treated with isotype-matched mAb (C) or anti-CD157 (D) are indicated. Control neutrophils reach the junction, stop, and begin to cross it. Neutrophils treated with anti-CD157 mAb move at least twice the distance of control neutrophils before reaching the site of diapedesis. In each experiment, neutrophils were plated on confluent HUVEC monolayers, and frames were captured every 10 seconds over a period of 40 minutes (bar represents 10 μm). Frames were then combined in stacks and exported in AVI format as movies. All the images were processed using the TrackIt! software (Olympus Biosystems). To measure the trajectory length of each migrating neutrophil, the cell centroid was tracked from the moment of cell-to-cell contact to the beginning of diapedesis (Figure S2). Micrographs are representative of 5 independent experiments. For a complete set of images, see Videos S1, S2, and S3 online.