Figure 3.
Effect of CSF-1 on RANKL and osteoclast marker expression in primary bone marrow cultures. Femurs were flushed, and bone marrow cells were plated at a density of 2.5 × 106 cells/cm2. Cells were incubated in complete medium alone (control) or in differentiation medium (10−8 m VD3/10−8 m DEX) and incubated in the presence and absence of rmCSF-1 (100 ng/ml). In some experiments, rmRANKL (50 ng/ml) was added to cultures. After 13 d, cultures were harvested for total RNA or TRACP assays. A, RT-PCR for RANKL and CTR mRNA expression. B and C, Semiquantitative analysis of RANKL and CTR expression shown in A. D and E, Effect of CSF-1 on TRACP activity. TRACP activity was assayed using p-nitrophenyl phosphate as a substrate, and the OD at 405 nm was determined by spectrophotometry. Osteoclasts (arrows) were identified by positive TRACP staining according to manufacturer’s protocol. F, Effect of CSF-1 concentrations on RANKL and osteoclastogenesis. Experiments were carried out as described in A using indicated doses of rmCSF-1. RT-PCR for RANKL and CTR expression was performed (left panel), and the number of TRACP-positive multinucleated cells (MNCs, defined as cells with at least three nuclei) per well was determined (right panel). a, P < 0.001; b, P < 0.01; c, P < 0.05 vs. 10−8 m VD3/10−8 m DEX-treated cultures.