Fig. 10. Transmigration of freshly isolated human monocytes through human brain endothelial cells is prevented by anti-PrP^C antibodies. (A) hCMEC/D3 cells were grown to confluence on Transwell filters with 3 µm pore size and activated with TNF- (200 U/ml) and IFN- (200 U/ml) for 24 hours. Freshly isolated and purified monocytes were labeled with CMFDA and were then added and allowed to transmigrate for 3 hours in the presence of MCP-1 (25 ng/ml) in basal compartments, following separate incubation of activated hCMEC/D3 cells and monocytes with the indicated antibodies at 20 µg/ml for 1 hour. Antibodies tested were specific for: the irrelevant membrane protein CD71; PECAM-1 (HEC-7 antibody), known to support monocyte transmigration; or PrP^C (SAF34 antibody). Average values (± s.e.m.) are presented of triplicates from three independent experiments with 100% being the number of transmigrated monocytes following incubation with anti-CD71 antibodies (*P<0.01 versus cells pre-treated with anti-CD71 antibody). (B) Migration of CMFDA-labeled monocytes was performed through endothelial-cell-free filters coated with fibronectin and collagen, for 3 hours in the presence of MCP-1 (25 ng/ml) in basal compartments. Cells were pre-incubated with the indicated antibodies at 20 µg/ml for 1 hour before the migration assay as above, with anti-CD71, anti-PECAM-1 (HEC-7) or anti-PrPC (SAF34) antibodies. The number of migrated cells following incubation with CD71 antibodies was taken as 100%. Average values (± s.e.m.) from one representative experiment out of three independent experiments performed in triplicate are presented. No significant difference was observed between the different conditions, indicating that pre-incubation with the indicated antibodies did not differentially affect the migration capacity of monocytes.