This study investigates osteoclastogenesis, focusing on the role of the anti-c-fms antibody in inhibiting the differentiation of osteoclast precursors. The methodology involves culturing mouse bone marrow cells and assessing the effects of various concentrations of the antibody on osteoclast formation.
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.
Seed 10 million cells per 10 milliliters in a 10-centimeter culture dish, and add M-CSF to the cells. Incubate the culture at 37 degrees Celsius, 5% carbon dioxide for 3 days. Remove the medium after three days, and wash the cells twice, vigorously, with 10 milliliters of PBS to remove non-adherent cells.
Add 5 milliliters of room temperature 0.02% trypsin-EDTA in PBS, and incubate at 37 degrees Celsius, 5% carbon dioxide for 5 minutes. Thoroughly pipette the cells to detach them. Observe the culture under a microscope to make sure the cells have been detached and appear rounded and floating in media.
When the cells have been detached, inactivate the reaction by adding 5 milliliters of alpha-MEM. Collect the cells into a 50-milliliter conical tube and centrifuge at 300 x g for 5 minutes. After discarding the supernatant, wash with 5 milliliters of alpha-MEM and centrifuge again at 300 x g for 5 minutes. Resuspend the pellet in 10 milliliters of alpha-MEM.
Seed 1 million cells per 10 milliliters in a 10-centimeter culture dish, and add M-CSF. Incubate the culture at 37 degrees Celsius, 5% carbon dioxide for 3 days. Harvest the attached cells, which represent BMMs as osteoclast precursors after 3 days. Seed BMMs at 50,000 cells per 200 microliters of alpha-MEM in a 96-well plate, per well.
To each well, add desired stimuli, and then add M-CSF for a final concentration of 100 nanograms per milliliter. Add anti-c-fms antibody to each well. Incubate the plate at 37 degrees Celsius, 5% carbon dioxide for 4 days, while changing media every other day for 4 days, and then proceed with staining and assays as described in the manuscript.
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