Protocol for the isolation of inducible GαsR201C explant cultures for ex vivo modeling of fibrous dysplasia of bone and pathologic osteoclastogenesis.

Fibrous dysplasia (FD) is caused by activating mutations in GNAS that disrupt skeletal progenitor differentiation and promote osteoclast-mediated bone resorption. Here, we describe a protocol to isolate bone marrow stromal cells from doxycycline-inducible Gαs∧R201C mice and assess osteoclast formation using tartrate-resistant acid phosphatase (TRAP) staining. We also detail procedures for confirming transgene induction by X-gal staining to detect β-galactosidase activity. This workflow provides a reproducible ex vivo platform to evaluate stromal-driven osteoclastogenesis in a controlled setting. For complete details on the use and execution of this protocol, please refer to Whitlock et al.1.