Encyclopedia of Experiments: Immunology
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Transcript
Osteoclastogenesis is the process by which osteoclast precursors differentiate and fuse to form multinucleated osteoclasts.
To study the effect of the anti-c-fms antibody on osteoclastogenesis in vitro, take a culture plate containing a mouse bone marrow cell suspension. Add macrophage colony-stimulating factor, M-CSF.
In culture, M-CSF binds to its specific receptor, c-fms, on myeloid precursor cells, activating signaling pathways and causing differentiation into bone marrow macrophages, BMMs, which represent osteoclast precursors and adhere to the plate.
Remove the non-adherent cells. Harvest the BMMs, and seed them onto multi-well plate wells. Supplement with M-CSF and receptor activator of nuclear factor kappa-Β ligand, RANKL.
Add increasing anti-c-fms antibody concentrations to the wells.
In the control well, M-CSF binds to the c-fms receptors and activates signaling pathways, stimulating RANK receptor expression and promoting BMM survival and proliferation. RANKL binds to the RANK receptors, triggering a signaling cascade and causing BMM differentiation and fusion into osteoclasts.
In wells with higher antibody concentrations, the antibodies bind and effectively block the c-fms receptors and disrupt the signaling required for osteoclast differentiation, thereby inhibiting osteoclast formation.
Perform staining to identify and quantify the osteoclast populations and determine the effective antibody concentration to inhibit osteoclastogenesis in RANKL-induced osteoclast formation.
