Effects of GM-CSF and cytokines on the growth of high-passage-number RA FLSs. (a) Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) 2–14 (at passage 11) were seeded at 1.5 × 104 cells per well in triplicate in a 24-well plate. Cells cultured in high-glucose DMEM supplemented with 10% FBS were treated with cytokines (each at 100 ng/ml) or 10 or 100 ng/ml granulocyte – macrophage colony-stimulating factor (GM-CSF) on day 0. Cells were harvested every 2 days and counted. (b) The growth restoration of RA FLSs mediated by synovial fluid (SF) was markedly inhibited by neutralizing antibody against GM-CSF. RA FLSs (2–6 and 2–14) at passage 12 were cultured at 5 × 103 cells per well in a 24-well plate. FLSs in culture were treated with GM-CSF (10 or 100 ng/ml) or SF at 1/10 dilution every 2 days for 6 days. SF-treated FLSs were cultured in the presence or absence of anti-GM-CSF neutralizing antibody (300 ng/ml). Cells were counted and assessed for viability by trypan blue staining every 2 days. The results are means ± SD obtained from single experiments performed in triplicate cultures. *p < 0.01; **p < 0.05.