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This protocol includes dissection of murine long bones and bone marrow isolation, to generate bone marrow macrophages and produce osteoclasts using macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL) or tumor necrosis factor alpha (TNF-α) and their arrest by anti-c-fms antibody, M-CSF receptor.
Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by osteoclasts in response to different stimuli. Osteoclast precursors differentiate into multinuclear osteoclasts in response to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL). Under pathologic conditions, the cytokine profile is different and involves a mixture of inflammatory cytokines. Tumor necrosis factor alpha (TNF-α) is one of the most important cytokines as it is found in large amounts in areas involved with inflammatory osteolysis. The purpose of this protocol is to provide a method by which murine bone marrow is isolated to generate osteoclasts through induction with M-CSF and either RANKL or TNF-α which will be subsequently inhibited by increasing doses of anti-c-fms antibody, the receptor for M-CSF. This experiment highlights the therapeutic value of anti-c-fms antibody in diseases of inflammatory bone resorption.
Osteoclasts are highly specialized cells, and they differentiate from hematopoietic stem cells through the fusion of multiple osteoclast precursors. They are essential for healthy bone remodeling and contribute to pathologic bone resorption associated with inflammatory osteolytic diseases such as rheumatoid arthritis and periodontal disease1.
Osteoclastogenesis and osteoclast function are mediated by two key factors; macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Both M-CSF and RANKL are important for osteoclast differentiation2. A....
All animal procedures and animal care were performed according to Tohoku University rules and regulations.
1. Murine Hindlimb Dissection and Handling
The purpose of this protocol is to evaluate the effect of anti-c-fms antibody on osteoclast formation in the presence of RANKL or TNF-α, and to determine the effect of M-CSF on osteoclast precursors proliferation. In this protocol, we have provided a reliable process by which large quantities of pure osteoclast cultures are generated. We have also provided a way to test anti-c-fms antibody suitable concentration for inhibiting osteoclast formation under different culture conditions.<.......
In this study, we investigated the effect of anti-c-fms antibody on RANKL-induced osteoclast formation, TNF-α-induced osteoclast formation and M-CSF-induced osteoclast precursor proliferation. We found that the effective amount of anti-c-fms antibody for the inhibition of osteoclastogenesis among RANKL-induced osteoclast formation, TNF-α induced osteoclast formation and M-CSF-induced proliferation of osteoclast precursors is different.
RANKL mediates osteoclastogenesis in the presenc.......
This work was supported in part by a JSPS KAKENHI grant from the Japan Society for the Promotion of Science (No. 16K11776 to H. K., No. 17K17306 to K. S., No. 16K20637 to K. K., No. 16K20636 to M. S., No. 18K09862 to I. M.).
....Name | Company | Catalog Number | Comments |
Anti-c-Fms antibody | AFS98, a rat monoclonal, antimurine, c-Fms antibody (IgG2a) | ||
TNF-α | Recombinant murine TNF-α prepared in our laboratory. TNF-α cDNA fragment cloned by RT-PCR and cloned into a pGEX-6P-I (Amersham Biosciences, Piscataway, NJ) to generate a GST-fusion protein. GST-TNF-α was expressed in Escherichia coli BL21 cells cells (Stratagene, La Jolla, CA). The cells were lysed under nondenaturing conditions and GST-TNF-α was purified over a glutathione-Sepharose column. GST was cleaved off by PreScission Protease (Amersham Biosciences) by manufacturer’s directions and was removed by a glutathione-Sepharose column. | ||
RANKL | PEPROTECH | 315-11 | Recombinant Murine sRANK Ligand, Source: E.coli |
M-CSF | Recombinant human M-CSF. 1/10 vol of CMG14–12 cell line culture supernatant at 5X106 cells in a 10-cm suspension culture dish. | ||
α-MEM | Wako | with L-Glutamine and phenol red | |
Fetal Bovine Serum | Biowest | s1820-500 | Fetal Bovine Serum French Origin |
Culture dish | Corning | 100 mm x 20 mm style dish | |
96-well plate | Thermofischer Scientific | Nun clon Delta surface | |
Cell counting kit-8 | Dojindo, Kumamoto, Japan | ||
Microplate reader | Sunrise REMOTE; Tekan Japan, Kawasaki, Japan | ||
Cell strainer | Corning | 40 μm Nylon | |
Centrifuge tube | Corning | 50 mL CentriStar cap | |
Triton X-100 | Wako | Polyoxyethylene (10) Octyphenyl ether |
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