Name: A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation (Video) | JoVE Journal
Uploaded: 2018-03-15T12:30:00.000+00:00
Duration: 9 min 37 s
Description: Learn a RANKL-based osteoclast culture assay using mouse bone marrow to investigate mTORC1's role in osteoclast formation. This method generates giant osteoclasts within one week for bone biology research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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
<ol>
	<li>Generate osteoclast specific Raptor deletion mice (Raptorfl/fl; Ctsk-cre, hereafter RapCtsk) by mating Raptorfl/fl mice with Ctsk-cre mice. Use the Raptorfl/fl mice as the WT control in this study.</li>
	<li>Have a container with ice to keep isolated bone.</li>
	<li>Prepare culture medium.
	<ol>
		<li>Prepare &#945;-MEM culture medium by supplementing minimum essential medium alpha (&#945;-MEM) with 1x glutamine, penicillin-streptomycin, and 10% fetal calf serum.</li>
		<li>Prepare bone marrow macrophage induction medium consisting of 50 mL of &#945;-MEM medium and M-CSF at 20 ng/mL.</li>
		<li>Prepare osteoclast induction medium consisting of M-CSF and RANKL at 20 ng/mL, respectively, in 50 mL of &#945;-MEM medium.</li>
	</ol>
	</li>
	<li>Prepare the following buffers before TRAP staining.
	<ol>
		<li>Prepare fix solution consisting of 6.5 mL of acetone, 2.5 mL of citrate solution and 0.8 mL of 37% (vol/vol) formaldehyde solution.</li>
		<li>Prepare TRAP staining solution.
		<ol>
			<li>Prewarm deionized water to 37 &#176;C.</li>
			<li>Add 100 &#181;L of fast garnet GBC base solution and 100 &#181;L of sodium nitrite solution into a 1.5-mL microtube and mix by gentle inversion for 30 s. Leave the mixture to stand for 2 min.</li>
			<li>Prewarm 9 mL of deionized water to 37 &#176;C. Add 200 &#181;L of diazotized fast garnet GBC base solution from step 1.4.2.2, 100 &#181;L of naphthol AS-BI phosphate solution, 400 &#181;L of acetate solution, and 200 &#181;L of tartrate solution.</li>
			<li>Keep the TRAP staining mixture solution in a water bath at 37 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare the following buffer before TRAP activity quantification of culture medium.
	<ol>
		<li>Prepare tartrate buffer (50 mL): 2 mL of 0.33 M L-tartaric acid, 48 mL of 0.33 M sodium tartrate dibasic dehydrate. Adjust the pH value to 4.9.</li>
		<li>Prepare 4-Nitrophenyl phosphate disodium (pNPP) buffer (200 mL): 1.502 g of glycine, 41 mg of MgCl2, 27.2 mg of ZnCl2, and 180 mL of deionized water. Adjust the pH value to 10.4 with 3 M NaOH and the volume to 200 mL with deionized water.</li>
		<li>Prepare pNPP buffer with phosphatase substrate (for one 96-well plate): 49.37 mg of phosphatase substrate and 175 &#181;L of pNPP buffer of step 1.5.2.</li>
		<li>Prepare substrate buffer (9 mL/96-well plate): 6.24 mL of deionized water, 360 &#956;L of acetate solution, and 2.4 mL of tartrate buffer. Mix by vortex and preheat at 37 &#176;C before use.</li>
		<li>Add 90 &#956;L of pNPP buffer with phosphatase substrate(1.5.3) to 9ml of preheated substrate buffer(1.5.4) to make a substrate mixture and vortex for 10 s.</li>
	</ol>
	</li>
</ol>
2. Dissection (Day -1)
<ol>
	<li>Euthanize 3 one-month-old female WT and RapCtsk mice by CO2 separately. Perform CO2 inhalation with appropriate equipment by trained personnel.</li>
	<li>Immerse 6 mice in a beaker with 100 mL of 75% ethanol (vol/vol) for 5 min to prevent bacterial contamination and then place on a dissection board in a supine position. Make an incision at the distal of the tibia vertically and dissect the skin along the hind limb with ophthalmic scissors.</li>
	<li>Cut the articular ligament of the hip joint with scissors and dislocate the hind limbs from the trunk.</li>
	<li>Cut the articular ligament of the knee joint and carefully disassociate the tibia and femur from the knee joint. Gently remove the soft tissue with scissors.</li>
	<li>Place all bones of one genotype in of one mouse in each well of a six-well-plate with 2 mL of &#945;-MEM on ice. 
	NOTE: To preserve cell viability, the bone should be kept in these conditions for less than 1 h.</li>
</ol>
3. Isolation (Day -1)
<ol>
	<li>Fill one well of a six-well plate with 75% ethanol (3 mL) and the other 5 wells with &#945;-MEM medium (3 mL).</li>
	<li>Put all bones of one genotype in the ethanol wash for 15 s and wash bones 5 times with &#945;-MEM medium for 10 s each time.</li>
	<li>Cut off the epiphyses with the scissor and insert a 0.45-mm syringe needle into the bone cavity and flush marrow out with &#945;-MEM medium into a 50-mL centrifuge tube.</li>
	<li>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.</li>
	<li>Centrifuge the bone marrow to obtain a cell pellet at 800 x g and 4 &#176;C for 5 min. Aspirate the supernatant.</li>
	<li>Add 3 mL of red blood cell lysis buffer into the centrifuge tube and pipette gently to resuspend the bone marrow. Keep the centrifuge tube on ice for 8 min to lyse red blood cells.</li>
	<li>Add 6 mL of &#945;-MEM medium into the centrifuge tube to stop cell lysis. Centrifuge at 500 x g and 4 &#176;C for 10 min and aspirate the supernatant.</li>
	<li>Resuspend in 3 mL of &#945;-MEM culture medium and place the cells into a six-well plate (generally one mouse per well).</li>
	<li>Incubate at 37 &#176;C in a 5% CO2 incubator overnight.</li>
</ol>
4. Plating (Day 0)
<ol>
	<li>Transfer the supernatant to a 15 mL centrifuge tube to collect the unattached cells the next morning.</li>
	<li>Centrifuge at 800 x g and 4 &#176;C for 5 min and aspirate the supernatant.</li>
	<li>Resuspend in 4 mL of &#945;-MEM culture medium.</li>
	<li>Take 20 &#181;L of the cell solution and mix with 20 &#181;L of Trypan Blue. Add 10 &#181;L of the mixture to the counting chamber and obtain the cell count per mL of solution.</li>
	<li>Add an appropriate volume of &#945;-MEM culture medium to obtain a cell solution of 500,000 cells/mL.</li>
	<li>Add 500 &#181;L and 50 &#181;L of bone marrow macrophage induction medium into each well of a 24-well plate and 96-well plate, respectively.</li>
	<li>Add 500 &#181;L and 50 &#181;L of cell solution into each well of a 24-well plate and 96-well plate, respectively.</li>
	<li>Incubate at 37 &#176;C in a 5% CO2 incubator for 3 days.</li>
</ol>
5. Differentiation (Days 3-7)
<ol>
	<li>Three days after plating, collect 50 &#181;L medium from each well of the 96-well-plate and freeze at -20 &#176;C, and then aspirate the residual medium.</li>
	<li>Add 1 mL and 100 &#181;L osteoclast induction medium into each well of a 24-well plate and a 96-well plate, respectively.</li>
	<li>Incubate at 37 &#176;C for 2 days.</li>
	<li>Collect 50 &#181;L of medium from each well and aspirate the residual medium.</li>
	<li>Add osteoclast induction medium into each well.</li>
	<li>Incubate at 37 &#176;C for 1 day. 
	NOTE: At this time point, large, multinucleated osteoclasts should be seen under an inverted microscope (Figure 2A).</li>
	<li>If osteoclasts are not seen, change the osteoclast induction medium and observe daily until osteoclasts are seen.</li>
	<li>Collect 50 &#181;L of medium from each well of the 96-well plate after osteoclasts have formed.</li>
</ol>
6. Tartrate Resistant Acid Phosphatase (TRAP) Staining
NOTE: Mature osteoclasts may be present after 6-7 days of in vitro culture (step 4).
<ol>
	<li>Aspirate medium and wash gently 3 times with 1x PBS.</li>
	<li>Fix the cells in each well of the 96-well plate with 100 &#181;L of fix solution for 30 s at room temperature.</li>
	<li>After fixation, aspirate the fix solution and wash gently 3 times with deionized water prewarmed to 37 &#176;C. Aspirate deionized water.</li>
	<li>Add 100 &#181;L of TRAP stain into each well of the 96-well plate and place the plate in an incubator at 37 &#176;C for 30 min and shield from light.</li>
	<li>After staining, aspirate the TRAP stain and gently wash 3 times with deionized water.</li>
	<li>Image osteoclasts using an inverted microscope at 40X magnification.</li>
</ol>
7. TRAP Activity Quantification of Culture Medium
<ol>
	<li>Collect 30 &#181;L culture supernatant from each well of the 96-well plate from step 5.1, 5.4 and 5.8; Use 30 &#181;L of non-cultured osteoclast induction medium as the negative control. Place the samples in a new 96-well plate.</li>
	<li>Add 90 &#956;L of substrate mixture(1.5.5) into each well of the 96-well plate.</li>
	<li>Place the plate in an incubator at 37 &#176;C for 1 h and shield from light.</li>
	<li>Stop the reaction by adding 50 &#181;L of 3 M NaOH into each well of the 96-well plate.</li>
	<li>Measure theabsorbance at the wavelength of 405 nm.</li>
</ol>
8. Western Blotting
NOTE: After 6-7 days of in vitro culture in the 24-well plate, mature osteoclasts should be visible.
<ol>
	<li>Aspirate the medium.</li>
	<li>Wash gently with pre-cooled 1x PBS 3 times.</li>
	<li>Aspirate PBS.</li>
	<li>Add 100 &#181;L of 1x SDS lysis buffer containing protease inhibitor cocktail.</li>
	<li>Heat lysates at 105 &#176;C for 10 min and centrifuge at 12,000 &#215; g and 4 &#176;C for 10 min. Collect the supernatants containing proteins.</li>
	<li>Separate the lysates containing 30 &#181;g of protein by 10% SDS-PAGE11 and then transfer to a polyvinylidene fluoride (PVDF) membrane.</li>
	<li>Block the membranes with 5% nonfat milk for 30 min.</li>
	<li>Incubate the membranes with anti-Raptor, anti-P-S6, anti-S6, anti-&#946;-actin at 1:1,000 dilution in 5% nonfat milk at 4 &#176;C overnight.</li>
	<li>Wash the membranes with Tris Buffered Saline with Tween-20 (TBST) for 10 min at room temperature.</li>
	<li>Repeat step 8.9 twice.</li>
	<li>Incubate the membranes with horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit IgG antibodies at 1:5,000 dilution in 5% nonfat milk for 1 h at room temperature.</li>
	<li>Wash the membranes 3 times with TBST for 10 min at room temperature.</li>
	<li>For chemiluminescent detection, add western chemiluminescent HRP substrate on the membranes and incubate for 5 min at room temperature. Drain the excess substrate and expose the blots to X-ray film.</li>
</ol>

<ol><li>Boyle, W. J., Simonet, W. S., Lacey, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osteoclast+differentiation+and+activation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Epigenetic+regulation+of+gene+expression%3A+how+the+genome+integrates+intrinsic+and+environmental+signals%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">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/pubmed?term=((Disorders+of+bone+remodeling%5BTitle%5D)+AND+%22Annu+Rev+Pathol%22%5BJournal%5D)">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/pubmed?term=((Advances+in+osteoclast+biology+reveal+potential+new+drug+targets+and+new+roles+for+osteoclasts%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">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/pubmed?term=((Identification+and+characterization+of+osteoclast-like+cells+and+their+progenitors+in+cultures+of+feline+marrow+mononuclear+cells%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Osteoprotegerin+ligand+is+a+cytokine+that+regulates+osteoclast+differentiation+and+activation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((TRANCE+is+a+novel+ligand+of+the+tumor+necrosis+factor+receptor+family+that+activates+c-Jun+N-terminal+kinase+in+T+cells%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((mTOR%3A+from+growth+signal+integration+to+cancer%2C+diabetes+and+ageing%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((The+two+TORCs+and+Akt%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">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/pubmed?term=((Inactivation+of+Regulatory-associated+Protein+of+mTOR+%28Raptor%29%2FMammalian+Target+of+Rapamycin+Complex+1+%28mTORC1%29+Signaling+in+Osteoclasts+Increases+Bone+Mass+by+Inhibiting+Osteoclast+Differentiation+in+Mice%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSambrook%2C+J+%22Molecular+cloning%3A+a+laboratory+manual%22">Molecular cloning: a laboratory manual</a>. , Cold spring harbor laboratory press. (1989).</li>
<li>Weischenfeldt, J., Porse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bone+Marrow-Derived+Macrophages+%28BMM%29%3A+Isolation+and+Applications%5BTitle%5D)+AND+%22CSH+Protoc%22%5BJournal%5D)">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/pubmed?term=((Osteoclast+culture+and+resorption+assays%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Osteoclast+derivation+from+mouse+bone+marrow%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((RANKL-based+osteoclastogenic+assays+from+murine+bone+marrow+cells%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Tumor+necrosis+factor+receptor+family+member+RANK+mediates+osteoclast+differentiation+and+activation+induced+by+osteoprotegerin+ligand%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((From+where+comes+the+osteoclast%3F%5BTitle%5D)+AND+%22J+Pathol%22%5BJournal%5D)">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/pubmed?term=((Control+of+bone+resorption+in+mice+by+Schnurri-3%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Control of bone resorption in mice by Schnurri-3.</a> Proc Natl Acad Sci U S A. 109 (21), 8173-8178 (2012).</li>
</ol>

All authors state that they have no conflicts of interest.

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>&lt;em&gt;Raptor&lt;sup&gt;fl/fl&lt;/sup&gt;&lt;/em&gt; mice</td><td>The Jackson Laboratory</td><td>013188</td><td></td></tr><tr><td>&lt;em&gt;Ctsk-cre&lt;/em&gt; mice</td><td></td><td></td><td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td></tr><tr><td>&amp;alpha;-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&amp;amp;D</td><td>Q3U4F9</td><td></td></tr><tr><td>Recombinant mouse RANKL protein</td><td>R&amp;amp;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&#039;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>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>ZnCl&lt;sub&gt;2&lt;/sub&gt;</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-&amp;beta;-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

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...

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 ...

a-rankl-based-osteoclast-culture-assay-mouse-bone-marrow-to

Research

JoVE Journal

Developmental Biology

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclast Derivation from Mouse Bone Marrow

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

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.

Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Learn a RANKL-based osteoclast culture assay using mouse bone marrow to investigate mTORC1's role in osteoclast formation. This method generates giant osteoclasts within one week for bone biology research.

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

Explore More Videos