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Fig. 8. Adhesion of U937 monocytic cells to human brain endothelial cells is not affected by anti-PrPC antibodies. (A) hCMEC/D3 cells were grown to confluence and activated (+) or not (–) with TNF-
(200 U/ml) and/or IFN-
(200 U/ml) for 24 hours. CMFDA-labeled U937 monocytic cells were then added and allowed to adhere for 30 minutes at 37°C. Numbers of adherent U937 cells are presented as percentages (%) of the total number of incubated cells. Average values (± s.e.m.) from one representative experiment out of three independent experiments performed in quadruplicate are presented (*P<0.1, **P<0.05 and ***P<0.01 versus non-activated cells: left-hand bar). (B) hCMEC/D3 cells were grown to confluence and activated with TNF- (200 U/ml) and IFN- (200 U/ml) for 24 hours; activated hCMEC/D3 cells and CMFDA-labeled U937 monocytic cells were separately incubated with the indicated antibodies at 20 µg/ml for 1 hour before the adhesion assay (30 minutes), as above. Antibodies tested were specific for the irrelevant membrane protein CD71, the junctional adhesion molecule not involved in leukocyte adhesion PECAM-1 (HEC-7 antibody), the monocyte integrin VLA-4 known to mediate monocyte adhesion, or PrPC (SAF32, SAF34, SAF61, 6H4 antibodies). Results are presented as percentages (%) of adherent U937 cells following incubation with the indicated antibodies, 100% being the number of adherent 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 quadruplicate are presented (*P<0.01).
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