Rs504393

Monocyte migration in response to chemokines and knee synovial fluid was tested using Transwell Permeable Supports (8 mm, Corning, USA). Circulating monocytes were plated in the upper chamber of 24-well plate inserts at a density of 1 Â 10 5 cells/well in serum-free medium. The next day, the cells were pretreated with a receptor antagonist (CCR1 antagonist BX471, CCR2 antagonist RS504393 and CCR5 antagonist TAK-779, all 100 nM; Medchem Express, USA) or 0.1% DMSO vehicle (control) for 1 h. Then, the cells were exposed to chemotaxis medium containing recombinant human CCL2, CCL3, CCL4 (all 10 ng/ml; R&D Systems, USA) or KOA synovial fluid (50% diluted in medium) in the presence or absence of the human CCL2, CCL3 and CCL4 neutralizing antibodies (all 1 mg/ ml; R&D Systems, USA). After 24 h, migrated monocytes in the lower chamber were fixed with 1% paraformaldehyde and stained with crystal violet dye (SigmaeAldrich, USA). The total number of migrated cells in the lower chamber was equal to the average number of cells in five random fields times the entire area of the lower chamber. The percent migration was calculated by dividing this number by the number of cells seeded.