Fig. 9. Transmigration of U937 monocytic cells 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 (+) or not (–) with TNF- (200 U/ml) and IFN- (200 U/ml) for 24 hours. CMFDA-labeled U937 monocytic cells were then added and allowed to transmigrate for 16-18 hours in the presence (+) or absence (–) of SDF-1 (100 ng/ml) in basal compartments. Transmigrated U937 cells were counted by FACS analysis of basal compartments. Results are presented as percentages of transmigrated cells. Average values (± s.e.m.) from one representative experiment out of three independent experiments performed in triplicate are presented. (B) The transmigration assay was performed as above in the presence of SDF-1 (100 ng/ml), following separate incubation of activated hCMEC/D3 cells and U937 monocytic cells 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 (SAF32, SAF34, SAF61, 6H4 antibodies). Average values (± s.e.m.) are presented of triplicates from three to five independent experiments with 100% being the number of transmigrated U937 cells following incubation with anti-CD71 irrelevant antibodies (*P<0.01 versus cells pre-treated with anti-CD71 antibody). (C) Migration of CMFDA-labeled U937 monocytic cells was performed through endothelial-cell-free filters coated with fibronectin and collagen, for 16-18 hours in the presence of SDF-1 (100 ng/ml) in basal compartments. Cells were preincubated with the indicated antibodies at 20 µg/ml for 1 hour before the migration assay, as above, with anti-CD71 or anti-PrP^C (SAF34, SAF32) antibodies. Results are presented as percentages (%) of transmigrated cells with 100% being the number of transmigrated U937 cells following incubation with anti-CD71 irrelevant antibodies. Average values (± s.e.m.) from one representative experiment out of three independent experiments performed in triplicate are presented. Numbers of migrated U937 cells following incubation with SAF32 or SAF34 anti-PrP^C antibodies were not statistically different from control values (migrated U937 cells following incubation with anti-CD71 antibodies), indicating that pre-incubation with the indicated antibodies did not differentially affect the migration capacity of U937 cells.