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.