Osteoclast formation in the presence of IFN-γ depends upon receptor-activator of NFκB ligand (RANKL) pretreatment in a dose-dependent and time-dependent manner. (a) RAW cells were pretreated with the indicated doses of RANKL for 2 days. On day 3, cells were switched to media with RANKL (200 ng/ml GST-RANKL) and various doses of IFN-γ, were then fixed and tartrate-resistant acid phosphatase (TRAP) stained after day 4. (b) The mean ± SEM of three independent experiments containing the highest dose of IFN-γ in (a) with statistics. *P = 0.05 compared with RANKL pretreatment at 0 ng/ml. (c) RAW cells treated continuously with a suboptimal dose of GST-RANKL (50 ng/ml), to slow osteoclastogenesis, were given IFN-γ (10 ng/ml) at the indicated time points. All cells were fixed and stained for TRAP on day 6. TRAP staining was quantitated densitometrically as described in Materials and methods. RANKL-treated RAW cells (no IFN-γ) stained on day 2 were only 10–20% TRAP-positive. All cultures with RANKL pretreatment ≥ 24 hours underwent osteoclastogenesis. *P = 0.05 compared with the IFN-γ day 0 group.