Expression of osteoclast markers on CFU-O derived colony cells in the c-kit^low population. (A) Cytospot samples prepared from day-14 agarose cultures were stained for α_vβ₃ vitronectin receptors. Note a large multinucleated cell showing positive reaction to anti-VTR. (B) Negative control stained for B220. (C) Cells from individual CFU-O–derived colonies cocultured for 7 days with ST2 cells in the presence of 1,25(OH)₂D₃ and hydrocortisone and then stained with anti-src. Note a group of cells expressing c-src (arrowheads) over the monolayer of ST2 cells in the background. (D) No positive staining on ST2 cells that were reacted with anti-src. (E) Autoradiography of cells from individual CFU-O–derived colonies cocultured for 14 days with ST2 cells in the presence of 1,25(OH)₂D₃and hydrocortisone and then exposed to ¹²⁵I-CT. Note a group of TRAPase-positive cells covered with dense silver grains (arrowheads) indicating the expression of calcitonin receptors. (F) Absence of ¹²⁵I-CT binding to cells incubated with an excess amount of cold CT. Scale bars represent 50 μm (A and B), 100 μm (C, E, and F), and 150 μm (D).