Induction of osteoclasts from human peripheral blood monocytes. (a) Purified CD16+ and CD16- peripheral blood monocytes were cultured with either macrophage colony-stimulating factor (M-CSF) (25 ng/ml) alone or with M-CSF (25 ng/ml) + receptor activator of NF-κB ligand (RANKL) (40 ng/ml) for 7 days and were stained for tartrate-resistant acid phosphatase (TRAP) activity. Original magnification, ×100. (b) The number of TRAP-positive multinucleated cells (MNC) (three or more nuclei) that differentiated from each monocyte subset was counted. (c) Resorbtive activity was assessed by culturing monocytes on plates coated with calcium phosphate films. The cells were treated with M-CSF (25 ng/ml) and RANKL (40 ng/ml) for 7 days. Arrows show resorbed lacunae. Original magnification, ×100. (d) Culture supernatants of CD16+ and CD16- were collected on day 7, and the concentrations of TRAP-5b and MMP-9 were measured with an ELISA. Representative data of more than three independent experiments are shown. Data represent the mean ± standard error of the mean values of duplicate or triplicate wells. *P < 0.01. Scale bars = 100 μm.