effect of the anti c-fms antibody on osteoclastogenesis in mouse bone marrow cells in vitro

Effect of the Anti C-fms Antibody on Osteoclastogenesis in Mouse Bone Marrow Cells In Vitro

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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1. Generation of BMM
<ol>
	<li>Seed 1 x 107 cells/10 mL in a 10 cm culture dish and add M-CSF 100 ng/mL. The final concentration of M-CSF is 100 ng/mL of culture medium. Incubate the culture at 37 &#176;C, 5% CO2 for 3 days.</li>
	<li>After 3 days, remove the culture medium, wash the cells vigorously with 10 mL of phosphate-buffered saline (PBS) twice to remove non-adherent cells, add 5 mL of room temperature 0.02% trypsin-EDTA in PBS, and incubate at 37 &#176;C, 5% CO2 for 5 min.</li>
	<li>Detach the cells by thorough pipetting. Make sure that the cells have been detached by observing the culture under a microscope (the cells should appear rounded and floating in media). If the cells remain attached, repeat pipetting thoroughly to detach them. When the cells have been detached, add 5 mL of &#945;-MEM to inactivate the reaction.</li>
	<li>Collect the cells into a 50 mL conical tube and centrifuge at 300 x g for 5 min. Discard the supernatant, and wash with 5 mL of &#945;-MEM.</li>
	<li>Centrifuge at 300 x g for 5 min and resuspend the pellet in 10 mL of &#945;-MEM.</li>
	<li>Perform a cell count.</li>
	<li>Seed 1 x 106 cells/10 mL in a 10 cm culture dish and add M-CSF 100 ng/mL. The final concentration of M-CSF is 100 ng/mL of culture medium. Incubate the culture at 37 &#176;C, 5% CO2 for 3 days.</li>
</ol>
2. Generation of Osteoclasts from BMM as Osteoclast Precursors
<ol>
	<li>After 3 days, harvest the attached cells which represent BMM as osteoclast precursors, following steps 1.2-1.7.</li>
	<li>Seed BMM at 5 x 104 cells/200 &#956;L of &#945;-MEM in a 96-well plate. Add RANKL for a final concentration of 50 ng/mL or TNF-&#945; for a final concentration of 100 ng/mL. Add M-CSF for a final concentration of 100 ng/mL to each well.</li>
	<li>Add anti-c-fms antibody (final concentrations of 0, 1, 10, 100, 1000 ng/mL) to each well.</li>
	<li>Incubate at 37 &#176;C, 5% CO2 for 4 days. Change media every other day for 4 days.</li>
</ol>
3. Tartrate-resistant Acid Phosphatase (TRAP) Stain
<ol>
	<li>After 4 days of incubation, gently aspirate the culture medium of each well. Wash each well once with 200 &#956;L of room temperature 1x PBS.</li>
	<li>Add 200 &#956;L of 10% formalin to each well for fixation and incubate for 1 h at room temperature. Aspirate the formalin and wash each well 3 times with deionized water.</li>
	<li>Add 200 &#956;L of 0.2% Triton X-100 in PBS to each well for permeabilization and incubate for 1 h at room temperature. Aspirate the Triton X-100 and wash each well 3 times with deionized water.</li>
	<li>Add 200 &#956;L of TRAP stain solution to each well. Visualize the stain under the microscope. It will take from 10-30 min for the stain to develop properly.</li>
	<li>Aspirate the stain and wash each well with deionized water 3 times, and air dry. Keep it at room temperature to use in further observation.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-c-Fms antibody</td>
			<td></td>
			<td></td>
			<td>AFS98, a rat monoclonal, antimurine, c-Fms antibody (IgG2a)</td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>PEPROTECH</td>
			<td>315-11</td>
			<td>Recombinant Murine sRANK Ligand, Source: E.coli</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td></td>
			<td></td>
			<td>Recombinant human M-CSF. 1/10 vol of CMG14&#8211;12 cell line culture supernatant at 5X106 cells in a 10-cm suspension culture dish.</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Wako</td>
			<td></td>
			<td>with L-Glutamine and phenol red</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>s1820-500</td>
			<td>Fetal Bovine Serum French Origin</td>
		</tr>
		<tr>
			<td>Culture dish</td>
			<td>Corning</td>
			<td></td>
			<td>100 mm x 20 mm style dish</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Thermofisher Scientific</td>
			<td></td>
			<td>Nun clon Delta surface</td>
		</tr>
	</tbody>
</table>

Source: Marahleh, A., et al. <a href="https://app.jove.com/v/59089">Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the effect of different anti-c-fms antibody concentrations on osteoclastogenesis, which is the process of osteoclast formation from osteoclast progenitors.

Source: Marahleh, A., et al. Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro. J. Vis. Exp. 
...

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-&#914; ligand, RANKL.
Add increasing anti-c-fms antibody concentrations to the wells.
In the control well,&#160; 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.

31 조회수. 생쥐 골수 세포의 골수 형성에 대한 Anti C-fms 항체의 효과시험관 내

Effect of the Anti C-fms Antibody on Osteoclastogenesis in Mouse Bone Marrow Cells In Vitro

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Effect of the Anti C-fms Antibody on Osteoclastogenesis in Mouse Bone Marrow Cells In Vitro