Characterization of primary cultured osteoblasts. (A) May-Giemsa–stained cytospin of osteoblasts after in vitro culture for 5 days of calvarial cells of newborn mice, in the presence of 10−7 M PTH. (B, upper panel) Immunostaining of in vitro–cultured osteoblasts with IgG control or an osteopontin-specific antibody. (B, lower panel) Flow cytometric analysis of in vitro–cultured osteoblasts demonstrates no staining with antibodies to hematopoietic (CD45) or endothelial (PECAM-1, Pan-endo) cells, but does show staining with VCAM-1. (C) Flow cytometric analysis of osteoblast monolayers confirms the uniform presence of cell surface proteins known to be present on osteoblasts (Sca-1, CD61, and ICAM-1), but no staining with CD62L, CD40, CD69, or CD44.
