Rho GTPase Techniques in Osteoclastogenesis

Historically, in vitro culturing of primary osteoclasts involved co-culturing of mononuclear monocytes with bone marrow stromal cells, thereby providing the cytokines required for osteoclast formation and multinucleation. Since the identification and cloning of receptor activator of nuclear factor kappa B ligand (RANKL), culturing primary osteoclasts in vitro has become much simplified. It has become apparent that the actin cytoskeleton is extremely important for the osteoclast, not only in terms of structural support, but also for adhesion, polarization, and migration. Rho family GTPases are key regulators of the actin cytoskeleton. In this chapter, we describe simple techniques in culturing primary osteoclasts from murine bone marrow cells, evaluating the activation states of Rho GTPases in osteoclasts, measuring the migratory abilities of monocytes, and introducing proteins of interest into osteoclasts using the TAT construct.