Figure 1

Effect of stromal cell-derived factor 1 on osteoclast precursor migration and differentiation. (a) Wild-type bone marrow cells were cultured with PBS or macrophage colony-stimulating factor (M-CSF) for 3 days to generate osteoclast precursors (OCPs). Cells were stained with allophycocyanin-labeled anti-CD11b and Phycoerythrin-labeled anti-Gr-1 antibodies and were subjected to fluorescence-activated cell sorting analysis. Distributions of CD11b⁺ and Gr-1^-/lo cells are shown. Rectangle (CD11b⁺/Gr-1^-/lo fraction), the majority of cells with osteoclast forming potency. (b) Wild-type OCPs were labeled with calcein AM and were seeded in the upper chamber of a transwell dish, and various amounts of stromal cell-derived factor 1 (SDF-1) were added to the lower chamber. Percentage of migrated cells in the lower chamber determined by calcein intensity (left panel). Cells that migrated to the lower chamber were cultured with M-CSF and RANKL to form osteoclasts. Numbers of tartrate-resistant acid phosphatase-positive (TRAP⁺) cells per well was assessed (right panel). (c) OCPs were seeded in the upper chamber of a transwell with or without 100 ng/ml SDF-1 in the lower chamber for 3 hours. Nonmigrated cells from the upper chamber and migrated cells from the lower chamber were cultured with M-CSF and RANKL to form osteoclasts. TRAP staining was formed. Bar graphs, numbers of TRAP⁺ cells/well (left panel). Representative pictures show TRAP-stained osteoclasts formed from the lower chambers with or without SDF-1 (×10). (d) OCPs were cultured with M-CSF and RANKL plus SDF-1 (200 ng/ml) on bone slices for 9 days. Numbers of osteoclasts and resorption pits per slice were counted. Data are the mean ± standard error of the mean of four wells. Experiments were repeated three times with similar results. *P < 0.05 versus samples from PBS-treated cells.