Transendothelial migration assay

HUVECs were seeded at 10⁵ cells/well in the upper chamber of Transwell inserts (diameter, 6.5 mm; pore size, 5.0 μm; Corning), labeled with PKH67 green fluorescence, and cultured overnight in vascular cell basal medium with vascular endothelial growth factor (ATCC). On the next day, isolated bone marrow monocytes were labeled with PKH26 red fluorescent dye and added to the top of endothelial cells, and MCP-1 was added to the lower chamber of the wells to stimulate the migration of monocytes. In some experiments, the monocytes were preincubated with 200 μg/ml P5 (126.8 μm) peptide for 20 min. After 3-h incubation at 37 °C, the number of migrated cells was determined by a Leica confocal microscope, and the results were reconstructed and analyzed using IMARIS 8.0 software.