Fibroblast-like Synoviocyte Culture and Proliferation

FLS were isolated from WT or Batf knockout (KO) mice and cultured as described by Zhao et al. [23 (link)]. FLS of passages 4–8 were used for further analysis. Pure FLS (> 90% CD90⁺/< 1% CD14⁺) were identified by flow cytometry using antibodies against CD90 and CD14 (Abcam). FLS proliferation in culture was quantified by measuring bromodeoxyuridine (BrdU) incorporation [20 (link)]. Briefly, FLS cultured in a 96-well plate were treated with or without tumor necrosis factor (TNF)-α (100 ng/ml), and BrdU labeling was detected using the cell proliferation enzyme-linked immunosorbent assay (ELISA) BrdU kit (Roche). Proliferating cells in synovial sections were identified by detecting Ki67 using an antibody obtained from Abcam. The empty adenovirus (Ad-C) and BATF-expressing adenovirus (Ad-Batf) were as previously described [4 (link)]. FLS were infected with Ad-Batf and Ad-C at the indicated multiplicities of infection (MOI) for 2 h, washed, and maintained for 48 h before analysis.