Development of a method for the identification of receptor activator of nuclear factor-?B+ populations in vivo

Osteoclasts are induced by macrophage colony-stimulating factor-1 (CSF-1) and receptor activator of nuclear factor-?B (RANK) ligand (RANKL). Monocyte/macrophage lineages thought to be osteoclast precursors; however, such cells have not been fully characterized owing a lack tools for their identification. Osteoclast precursors express (CSF-1R) RANK. However, the capacity conventional methods using anti-RANK antibodies detect RANK+ flow cytometry is insufficient. Here, we developed high-sensitivity method detecting biotinylated recombinant glutathione S-transferase-RANKL (GST-RANKL-biotin). We sorted sub-populations mouse bone marrow (BM) or peripheral blood (PB) GST-RANKL-biotin, anti-CSF1R, anti-B220 osteoclastogenesis in vitro. The frequency population BM detected GST-RANKL-biotin was significantly higher than that antibodies. Although were both B220+ B220? populations, lineage present only B220?. Unexpectedly, number osteoclasts RANK?CSF-1R+ RANK+CSF-1R+ contained population. In contrast, PB-derived B220?RANK+CSF-1R+ B220?RANK?CSF-1R+ These results suggest useful detection RANK CSF-1R may helpful indicators PB.