The authors thank Dr. Minghan Tong and S. Kato for kindly providing reagents and mice. We thank the members of the Zou lab for useful discussions. This work was supported in part by grants from 973 Program from the Chinese Ministry of Science and Technology (MOST) [2014CB964704 and 2015CB964503], Clinical Research Program of 9th People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine. Thanks for the help of Core Facility for Cell Biology and Core Facility for Chemical Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

All procedures relating to the animals were performed according to the protocol approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC) and were approved by the Animal Care and Use Committee of the Shanghai Institute of Biochemistry and Cell Biology.
1. Preparation
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	<li>Generate osteoclast specific Raptor deletion mice (Raptorfl/fl; Ctsk-cre, hereafter RapCtsk) by mating Raptorfl/fl mic...

<ol>
	<li>Boyle, W. J., Simonet, W. S., Lacey, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+differentiation+and+activation.">Osteoclast differentiation and activation.</a> Nature. 423 (6937), 337-342 (2003).</li><li>Jaenisch, R., Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+gene+expression:+how+the+genome+integrates+intrinsic+and+environmental+signals.">Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals.</a> Nature Genetics. 33, 245-254 (2003).</li><li>Feng, X., McDonald, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+of+bone+remodeling.">Disorders of bone remodeling.</a> Annu Rev Pathol. 6, 121-145 (2011).</li><li>Boyce, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+osteoclast+biology+reveal+potential+new+drug+targets+and+new+roles+for+osteoclasts.">Advances in osteoclast biology reveal potential new drug targets and new roles for osteoclasts.</a> J Bone Miner Res. 28 (4), 711-722 (2013).</li><li>Ibbotson, K. J., Roodman, G. D., McManus, L. M., Mundy, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+osteoclast-like+cells+and+their+progenitors+in+cultures+of+feline+marrow+mononuclear+cells.">Identification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrow mononuclear cells.</a> J Cell Biol. 99 (2), 471-480 (1984).</li><li>Lacey, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoprotegerin+ligand+is+a+cytokine+that+regulates+osteoclast+differentiation+and+activation.">Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.</a> Cell. 93 (2), 165-176 (1998).</li><li>Wong, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRANCE+is+a+novel+ligand+of+the+tumor+necrosis+factor+receptor+family+that+activates+c-Jun+N-terminal+kinase+in+T+cells.">TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells.</a> J Biol Chem. 272 (40), 25190-25194 (1997).</li><li>Zoncu, R., Efeyan, A., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR:+from+growth+signal+integration+to+cancer,+diabetes+and+ageing.">mTOR: from growth signal integration to cancer, diabetes and ageing.</a> Nat Rev Mol Cell Biol. 12 (1), 21-35 (2011).</li><li>Bhaskar, P. T., Hay, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+TORCs+and+Akt.">The two TORCs and Akt.</a> Dev Cell. 12 (4), 487-502 (2007).</li><li>Dai, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Regulatory-associated+Protein+of+mTOR+(Raptor)/Mammalian+Target+of+Rapamycin+Complex+1+(mTORC1)+Signaling+in+Osteoclasts+Increases+Bone+Mass+by+Inhibiting+Osteoclast+Differentiation+in+Mice.">Inactivation of Regulatory-associated Protein of mTOR (Raptor)/Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Osteoclasts Increases Bone Mass by Inhibiting Osteoclast Differentiation in Mice.</a> J Biol Chem. 292 (1), 196-204 (2017).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (1989).</li><li>Weischenfeldt, J., Porse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+Marrow-Derived+Macrophages+(BMM):+Isolation+and+Applications.">Bone Marrow-Derived Macrophages (BMM): Isolation and Applications.</a> CSH Protoc. 2008, (2008).</li><li>Bradley, E. W., Oursler, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+culture+and+resorption+assays.">Osteoclast culture and resorption assays.</a> Methods Mol Biol. 455, 19-35 (2008).</li><li>Tevlin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+derivation+from+mouse+bone+marrow.">Osteoclast derivation from mouse bone marrow.</a> J Vis Exp. (93), e52056 (2014).</li><li>Xing, L., Boyce, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANKL-based+osteoclastogenic+assays+from+murine+bone+marrow+cells.">RANKL-based osteoclastogenic assays from murine bone marrow cells.</a> Methods Mol Biol. 1130, 307-313 (2014).</li><li>Hsu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor+receptor+family+member+RANK+mediates+osteoclast+differentiation+and+activation+induced+by+osteoprotegerin+ligand.">Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.</a> Proc Natl Acad Sci U S A. 96 (7), 3540-3545 (1999).</li><li>Underwood, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+where+comes+the+osteoclast?.">From where comes the osteoclast?.</a> J Pathol. 144 (4), 225-226 (1984).</li><li>Wein, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+bone+resorption+in+mice+by+Schnurri-3.">Control of bone resorption in mice by Schnurri-3.</a> Proc Natl Acad Sci U S A. 109 (21), 8173-8178 (2012).</li></ol>

The osteoclastogenic assay is the most widely used method to isolate and culture osteoclasts in vitro12,13. While several RANKL-based osteoclast inductions have been described13,14,15, the present study described a protocol with some modifications based on the previous methods.
In the previous study, BMMs were plating immediately afte...

Bone is an ever-changing organ and is remodeled by osteoblasts and osteoclasts throughout life. Osteoclasts are responsible for mineralized matrix resorption and osteoblasts synthesize and secrete new bone matrices1. The balance between bone resorption and bone formation is crucial for bone health including maintenance of bone mass and response to stimulation and injury. If this balance is disrupted, a series of bone metabolic diseases may occur, including osteoporosis and periodontal diseases. In these diseases, bone mass loss resulting from osteoclastic bone resorption exceeds the bone forming capacity of osteoblasts2,3. Thus, in order to develop pharmaceutical targets to treat skeletal disorders such as osteoporosis, it is critical to understand the generation and biology of osteoclasts4.
Osteoclasts are unique giant multinucleated cells located at or near the bone surface, and belong to the monocyte/macrophage family1. Ibbotson K. J. et al. reported a method to generate osteoclast-like cells in vitro with medium containing 1,25-dihydroxy-vitamin D35. The identification of macrophage-colony stimulating factor (M-CSF) and receptor activator for nuclear factor-&#954; B ligand (RANKL) as essential factors of osteoclast formation has dramatically increased the efficiency of osteoclastogenesis in vitro1,6,7. The ability to culture osteoclasts in vitro has improved our understanding of the generation and regulation of osteoclasts.
The mammalian/mechanistic target of rapamycin (mTOR) functions in two structurally and functionally distinct complexes, namely mTORC1 and mTORC28,9. The two multi-protein complexes are distinct from each other due to their different components and downstream substrates. mTORC1 contains the unique regulatory-associated protein of mTOR (Raptor), while mTORC2 contains the rapamycin-insensitive companion of mTOR (Rictor)9. mTORC1 can integrate and transmit important signals to regulate cell growth, proliferation and differentiation. Recently, we demonstrated that mTORC1 plays a key role in the network of catabolic bone resorption by deletion of Raptor to inactivate mTORC1 in osteoclasts10. However, the underlying mechanisms require further study. In the present study, a RANKL-based osteoclastogenic method was used to generate osteoclasts from bone marrow-derived macrophages (BMMs) of wild-type (WT) and RapCtsk mice, and to study the influence of mTORC1 inactivation on osteoclast formation.

<table>
	<tbody>
		<tr>
			<td>Raptorfl/fl mice</td>
			<td>The Jackson Laboratory</td>
			<td>013188</td>
			<td></td>
		</tr>
		<tr>
			<td>Ctsk-cre mice</td>
			<td></td>
			<td></td>
			<td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Corning</td>
			<td>10-022-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibico</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin streptomycin</td>
			<td>Gibico</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>BioInd</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse M-CSF protein</td>
			<td>R&#38;D</td>
			<td>Q3U4F9</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse RANKL protein</td>
			<td>R&#38;D</td>
			<td>Q3TWY5</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Beyotime</td>
			<td>C3702</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma-Aldrich</td>
			<td>302643</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate solution</td>
			<td>Sigma-Aldrich</td>
			<td>915</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Garnet GBC Base solution</td>
			<td>Sigma-Aldrich</td>
			<td>3872</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Nitrite Solution</td>
			<td>Sigma-Aldrich</td>
			<td>914</td>
			<td></td>
		</tr>
		<tr>
			<td>Naphthol AS-BI Phosphate Solution</td>
			<td>Sigma-Aldrich</td>
			<td>3871</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetate solution</td>
			<td>Sigma-Aldrich</td>
			<td>3863</td>
			<td></td>
		</tr>
		<tr>
			<td>Tartrate solution</td>
			<td>Sigma-Aldrich</td>
			<td>3873</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Corning</td>
			<td>21-031-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>L-tartaric acid</td>
			<td>Sigma-Aldrich</td>
			<td>251380</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tartrate dibasic dehydrate</td>
			<td>Sigma-Aldrich</td>
			<td>s4797</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase substrate</td>
			<td>Sigma-Aldrich</td>
			<td>P4744</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Raptor</td>
			<td>Cell Signaling Technology</td>
			<td>2280</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-P-ribosomal protein S6 (S235/236)</td>
			<td>Cell Signaling Technology</td>
			<td>2317</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ribosomal protein S6</td>
			<td>Cell Signaling Technology</td>
			<td>2211</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-&#946;-actin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-130300</td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde</td>
			<td>Xilong scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polyvinylidene fluoride (PVDF) membrane</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Western Chemiluminescent HRP Substrate (ECL)</td>
			<td>Millipore</td>
			<td>00000367MSDS</td>
			<td></td>
		</tr>
		<tr>
			<td>IX71</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Envision</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.45-mm Syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito forcep</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rankl-based osteoclast culture assay of mouse bone marrow to investigate the role of mtorc1 in osteoclast formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

Using the present protocol, a large number of giant osteoclasts were seen on day 6; if giant osteoclasts are not seen, one more day of osteoclast differentiation may be needed (Figure 1). Successful osteoclast formation was confirmed by TRAP staining (Figure 2A). Osteoclasts were giant wine red/purple cells with more than 3 nuclei. More than 250 osteoclasts were obtained in each well of the 96-well plate in WT BMMs (<strong class...

Watch this Scientific Journal Video about A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation at JoVE.com

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

The overall goal of this protocol is to isolate and culture osteoclasts in vitro from mouse bone marrow and to study the role of the mammalian target of rapamycin complex one in osteoclast formation. This method can help answer key questions in the formation of osteoclasts, such as the role of mTORC1 in osteoclast differentiation. The main advantage of this technique is to result in a large number of giant osteoclasts within one week.The implications of this technique extend toward the therapy of osteoporosis because it is helpful to study the mechanism by which osteoclast differentiates from their precursors. To begin this procedure, make a vertical incision at the distal end of the tibia of a euthanized mouse, and dissect the skin along the hind limbs. Next, cut the articular ligament of the hip joints with scissors, and dislocate the hind limbs from the trunk.Cut the articular ligament of the knee joint, and carefully disassociate the tibia and femur from it. Gently remove the soft tissue with scissors. Place all of the bones of one mouse in each well of a six-well plate with two milliliters of alpha-MEM on ice.For isolation, fill one well of a six-well plate with 75%ethanol and the other five wells with alpha-MEM. Put all the bones of one mouse in the ethanol wash for 15 seconds, and then wash the bones five times with alpha-MEM for 10 seconds each time. Cut off the epiphyses with the scissor, and insert a 0.45-millimeter syringe needle into the bone cavity.Flush the marrow out with alpha-MEM into a 50-milliliter centrifuge tube. Next, flush the bone cavity using the same method from the other end of the bone. Repeat at least twice to wash the bone cavity thoroughly until the bone is pale.Subsequently, centrifuge the bone marrow to obtain a cell pellet at four degrees Celsius for five minutes. Afterward, aspirate the supernatant. Add three milliliters of red blood cell lysis buffer into the centrifuge tube, and pipette gently to resuspend the bone marrow.Then, keep the centrifuge tube on ice for eight minutes to lyse the red blood cells. After eight minutes, add six milliliters of alpha-MEM into the centrifuge tube to stop the cell lysis. Centrifuge at 800 g and four degrees Celsius for 10 minutes, and aspirate the supernatant.Then, resuspend in three milliliters of alpha-MEM culture medium, and place the cells into a six-well plate. Incubate at 37 degrees Celsius overnight. In this step, transfer the supernatant to a 15-milliliter centrifuge tube to collect the unattached cells in the next morning.Afterward, centrifuge at 800 g and four degrees Celsius for five minutes, and aspirate the supernatant. Then, resuspend the cells in four milliliters of alpha-MEM culture medium. Take 20 microliters of the cell solution and mix with 20 microliters of Trypan Blue.Subsequently, add 10 microliters of the mixture to the counting chamber, and obtain the cell count per milliliter of solution. Following that, add an appropriate volume of alpha-MEM culture medium to obtain a cell solution of 500, 000 cells per milliliter. Next, add 500 microliters and 50 microliters of bone marrow macrophage induction medium into each well of a 24-well plate and 96-well plate, respectively.Afterward, add 500 microliters and 50 microliters of cell solution into each well of a 24-well plate and 96-well plate, respectively. Incubate at 37 degrees Celsius for three days. Three days after plating, collect 50 microliters of medium from each well of the 96-well plate, and freeze it at negative 20 degrees Celsius.Then, aspirate the residual medium. Add one milliliter and 100 microliters of osteoclast induction medium into each well of a 24-well plate and a 96-well plate, respectively. Incubate at 37 degrees Celsius for two days.After two days, collect 50 microliters of medium from each well, and aspirate the residual medium. Then, add osteoclast induction medium into each well, and incubate at 37 degrees Celsius for one day. After osteoclasts have formed, collect 50 microliters of medium from each well of the 96-well plate.In this step, aspirate the medium, and wash the sample gently three times with 1X PBS. Next, fix the cells in each well of the 96-well plate with 100 microliters of fix solution for 30 seconds at room temperature. After fixation, aspirate the fix solution, and wash the cells gently three times with deionized water prewarmed to 37 degrees Celsius.Then, aspirate the deionized water, and add 100 microliters of TRAP stain into each well of the 96-well plate. Place the plate in an incubator at 37 degrees Celsius for 30 minutes, and shield from light. After staining, aspirate the TRAP stain, and gently wash three times with deionized water.Subsequently, image the osteoclasts in an inverted microscope at 40X magnification. Then, transfer 30 microliters of the medium collected before to a new 96-well plate. Next, add 90 microliters of substrate mixture into each well of the 96-well plate.Place the plate in an incubator at 37 degrees Celsius for one hour, and shield from light. After an hour, stop the reaction by adding 30 microliters of three molar sodium hydroxide into each well of the 96-well plate. Then, measure the absorbance at the wavelength of 405 nanometers by the EnVision Multilabel Plate Reader.Here is the representative view of the osteoclasts observed in bright field on day six. The red line outlined the border of the osteoclasts. And shown here is a typically giant, multinucleated, TRAP-positive osteoclast.The black arrow indicated two nuclei of the multinucleated osteoclast. This figure shows TRAP staining of BMMs on day six. High-magnification images of the squares in wild-type and Ctsk-Cre Raptor knockout groups are shown, respectively.These were less giant, TRAP-positive osteoclasts formed in the Ctsk-Cre Raptor knockout BMMs in comparison to the wild-type group. This figure shows the TRAP activity of cultured wild-type and Ctsk-Cre Raptor knockout BMMs. Once mastered, this technique can be done within one week if it's performed properly.While attempting this procedure, it's important to remember to maintain the viability of the bone marrow macrophages, as the optimal seeding density of viable bone marrow macrophages is critical. It is recommended to keep the bone on ice no more than one hour and resuspend the cells gently. This procedure can be combined with other methods, such as the osteoclast resorption assay with bone slices, to study the normal and the pathological physiology of osteoclast.With this method, we found mTORC1 play an important role in osteoclast formation, which may act as a potential pharmacologic target for the treatment of bone metabolic disorders, such as osteoporosis, in the future.

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12.5K visualizações.  National Clinical Research Center of Stomatology. The overall goal of this protocol is to isolate and culture osteoclasts in vitro from mouse bone marrow and to study the role of the mammalian target of rapamycin complex one in osteoclast formation. This method can help answer key questions in the formation of osteoclasts, such as the role of mTORC1 in osteoclast differentiation. The main advantage of this technique is to result in a large number of giant osteoclasts within one week.The implications of this technique extend toward the...