Rapid,simple identification of individual osteoblastic cells and their specific products by cell blotting assay

Biochemical and molecular biological studies of osteoblastic cell function and hormonal regulation are frequently confounded by the inherent cellular heterogeneity and phenotypic instability of existing in vitro and in vivo model systems. A new technique (derived from Western blotting or antibody-based detection of protein molecules bound to nitrocellulose paper) is described for identification of individual cells which synthesize osteoblast-specific gene products (bone Gla-protein, type I collagen, and alkaline phosphatase) or produce cAMP in response to parathyroid hormone (PTH) or isoproterenol. Dispersed primary neonatal rat calvariae or osteogenic sarcoma cells were “plated” on Immobilon-P (a hydrophobic transfer membrane with very high protein-binding capacity) for 30 minutes to several hours, followed by agonist treatment, formalin fixation, hematoxylin staining, and immunostaining with a battery of antibodies specific for osteoblastic products. Individual cells and their secretory zones were visualized by light microscopy and counted. Treatment with PTH with or without isoproterenol resulted in increases in the percentages of osteoblastic cells elaborating cAMP, as well as the intensity of immunostaining, but had no effects on MCF-7 cells, a nonosteoblastic breast carcinoma control line. The percentage of cells within each primary osteoblastic cell population isolated or rat osteogenic sarcoma cell clone (G2 or C12) that elaborated bone-specific proteins or that generated cAMP in response to PTH varied with time and the individual cellular preparation, reconfirming the cellular heterogeneity of these systems. This method, in conjunction with techniques such as in vitro hybridization, should prove useful in characterizing discrete osteoblastic bone cell subpopulations and in clarifying mechanisms of hormonal regulation by local and systemic agents.