Cell culture
PBMCs were isolated from the blood of RA patients as described above. CD14+ monocytes were positively selected via magnetic-activated cell sorting (Miltenyi Biotec, Germany) and an anti-CD14 antibody. The isolated CD14+ monocytes (1.5 × 105 cells per well in 48-well plates or 6 × 105 cells per well in 12-well plates) were incubated in RPMI-1640 media containing 10% fetal bovine serum (FBS). Osteoclast differentiation was induced from CD14+ monocytes treated with macrophage colony-stimulating factor (M-CSF, 50 ng/ml) and receptor activator of nuclear factor kappa-Β ligand (RANKL, 100 ng/ml). C-X-C motif chemokine ligand 2 (CXCL2, 100 ng/ml or otherwise as indicated) were added to the cultures. The medium was replaced every 3 days.
RAW264.7 macrophages (1.0 × 104 cells per well in 48-well plates or 4.5 × 104 cells per well in 12-well plates) were incubated in DMEM media containing 10% FBS. Osteoclast differentiation was induced by M-CSF (10 ng/ml) and RANKL (50 ng/ml) with or without CXCL2.