Isolation of mouse osteocytes using cell fractionation for gene expression analysis.

Osteocytes are the terminally differentiated cells of the osteoblastic lineage embedded within the mineralized bone matrix. T: hey have been identified as key players in mechanotransduction and in mineral and phosphate homeostasis. In addition, they appear to have a role in mediating bone formation, since they secrete the bone formation inhibitor sclerostin. In contrast to osteoblasts and osteoclasts, which reside on the bone surface, it has been difficult to isolate and analyze cellular and molecular properties of osteocytes due to their specific location inside the "hard" mineralized bone compartment. This chapter describes a method to isolate osteocytes from newborn mouse calvaria and adult mouse long bone, followed by immediate total RNA extraction allowing to selectively study osteocytic versus osteoblastic gene expression by quantitative real-time polymerase chain reaction (qPCR). The osteocyte-enriched cell fraction isolated by this method can further be purified by FACS and selectively expresses osteocytic marker genes, such as Dmp1 and Sost.