We acknowledge the support of NIH grants R01 DE021683, R01 DE019434, U01 HL099776, The Oak Foundation and The Hagey Laboratory for Pediatric Regenerative Medicine.

NOTE: Ethical statement: All research involving vertebrate animals was performed in accordance protocols approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC).
1. Preparation
<ol>
	<li>Allow 10 ml of commercially available density gradient cell separation media (which contains polysucrose and sodium diatrizoate, adjusted to a density of 1.077 g/ml) to come to RT in a 50 ml conical tube.</li>
	<li>Prepare flow cytometry (FACS) buffer with 1x phosphate buffered saline (PBS) and 2% fetal calf serum (FCS). Take a 50 ml aliquot and maintain this aliquot at RT for use during the density gradient cell separation media step.</li>
	<li>Have a bucket of ice ready to maintain isolated tissue and cells in during the isolation procedure.</li>
</ol>
2. Prepare Culture Media
<ol>
	<li>Prepare basal media: MEM (Minimal Essential Medium), without phenol red (500 ml), glutamate (1x), FCS (10x), 10,000 units/ml penicillin (1x).</li>
	<li>Filter basal media with a 0.22 &#956;m filter.</li>
	<li>Prepare bone marrow macrophage induction media:
	<ol>
		<li>Take 50 ml of the basal media prepared in Step 2.1.</li>
		<li>Add Prostaglandin E2 (PGE2, Mr 352.465 g/mol) at 10-7 M final concentration.</li>
		<li>Add Macrophage-Colony Stimulating Factor (M-CSF) at 10 ng/ml.</li>
		<li>Do not filter the final solution of bone marrow macrophage stimulating media.</li>
	</ol>
	</li>
	<li>Prepare osteoclast induction media:
	<ol>
		<li>Take 50 ml of the basal media prepared in Step 2.1.</li>
		<li>Add Prostaglandin E2 (PGE2, Mr 352.465 g/mol) at 10-7 M final concentration</li>
		<li>Add Macrophage-Colony Stimulating Factor (M-CSF) at 10 ng/ml.</li>
		<li>Add RANKL at 10 ng/ml.</li>
		<li>Do not filter the final solution of osteoclast induction media.</li>
	</ol>
	</li>
</ol>
3. Bone Marrow Isolation
<ol>
	<li>Euthanize one C57BL/6 mouse using carbon dioxide inhalation method, followed by cervical dislocation. 
	NOTE: Rodents must be euthanized by trained personnel using appropriate technique, equipment and agents.</li>
	<li>Position and secure the mouse on a dissection board and spray with 70% alcohol.</li>
	<li>Dissect out the femurs, tibiae, humeri and spine.
	<ol>
		<li>Begin with the mouse in supine position. Make an incision vertically along the lower limb and begin by dissecting along the length of the femur and tibia by dividing soft tissue attachments. Finally, dislocate bones from the knee and hip joint to free bones fully from the skeleton.</li>
		<li>Proceed to make an incision along the length of the upper limb to isolate the humerus. Dislocate the humerus at the shoulder and elbow joints by dissecting soft tissue capsular attachment.</li>
		<li>Finally, turn the mouse over, so that the mouse lies prone. Make a skin incision along the length of the spine, in the midline. Observe the paravertebral soft tissue on each side of the spine and incise along the bony edge of the spine, dissecting through the paravertebral soft tissue mass. Using a scissors, make a horizontal cut across the spine at the base of the neck, and at the end of the spine, just proximal to the tail insertion, freeing the spine from soft tissue attachment.</li>
	</ol>
	</li>
	<li>Once the bones are harvested, keep them in FACS buffer on ice.</li>
	<li>Use tissue paper to clean muscle and soft tissue from the bones.</li>
	<li>Place the cleaned bones into a pestle and mortar with 3 ml of FACS buffer.</li>
	<li>Crush the bones gently in the pestle and mortar.</li>
	<li>Aspirate off the blood-stained fluid and transfer into a 50 ml conical tube by passing the solution through a 70 &#956;m cell strainer.</li>
	<li>Add 5 ml FACS buffer to the crushed bone and crush further. Again, remove the fluid using a pipette and transfer to the 50 ml conical tube through a 70 &#956;m strainer.</li>
	<li>Typically crush the bones twice to three times in fresh FACS buffer, using a mortar and pestle, each time adding a further 5 ml of fresh FACS buffer, until the fluid does not stain red.</li>
	<li>Centrifuge the bone marrow solution at 200 x g for 5 min at 4 &#176;C to yield a cell pellet.</li>
</ol>
4. Gradient Separation of Bone Marrow Cells
<ol>
	<li>Aspirate the supernatant and resuspend in 10 ml of FACS buffer (maintained at RT).</li>
	<li>Take the 50 ml conical tube containing 10 ml of commercially available density gradient cell separation media (RT).</li>
	<li>Layer the bone marrow solution onto the density gradient cell separation media. Do this slowly using an electric pipette. Angle the pipette tip against the inner surface near the top of the conical tube and tilt the conical tube to about 30&#176;. Using a slow speed setting on the pipette, gently expel the bone marrow solution so as not to disturb the density gradient cell separation media. 
	NOTE: Ultimately there will be a clear definition between the two solutions (cellular solution and density gradient cell separation media solution).</li>
	<li>Using a RT centrifuge, centrifuge the density gradient cell separation media and cell solution in a balanced centrifuge at 200 x g for 15 min. In addition, remove acceleration and deceleration on the centrifuge to ensure adequate phase separation using the density gradient cell separation media gradient.</li>
	<li>After centrifugation, aspirate off the cloudy middle layer which contains the bone marrow cells of interest.</li>
	<li>Transfer these cells into a new conical tube. Add 20 ml of additional FACS buffer (kept on ice) to wash the cells.</li>
	<li>Centrifuge at 200 x g for 5 min at 4 &#176;C to yield a cell pellet.</li>
	<li>Resuspend the final cell pellet in 1ml of macrophage stimulating media to allow for cell counting using a hemocytometer.</li>
</ol>
5. Cell Counting
<ol>
	<li>Take 10 &#181;l of the cell solution and mix with 10 &#956;l of trypan blue. Load 10 &#181;l of the resultant mix onto a hemocytometer and obtain the cell count per ml of solution.</li>
</ol>
6. Cell Culturing
<ol>
	<li>Place 2 ml of macrophage stimulating media into each well of a 24-well plate.</li>
	<li>Add 200,000 cells to each well. 
	NOTE: In our experience, cell-plating density is one of the most crucial requirements for adequate in vitro culturing of osteoclasts.</li>
	<li>Gently agitate the plate and place in a regular incubator at 37 &#176;C.</li>
	<li>Do not change the media for 3 days.</li>
	<li>After 3 days in macrophage stimulating media, change the media to osteoclast media.</li>
	<li>Hereafter, change the media daily for 5&#8211;7 days, at which point, large, multinucleated osteoclasts should be visible on the culture plate.</li>
</ol>
7. Staining with Tartrate Resistant Acid Phosphatase (TRAP)
<ol>
	<li>After 5&#8211;7 days of in vitro culture, aspirate the media from the culture well.</li>
	<li>Wash the wells gently with 1x PBS three times.</li>
	<li>Fix the cells by adding 4% paraformaldehyde solution and leave for 1 hr at 4 &#176;C.</li>
	<li>During this time, prepare the TRAP stain (which is commercially available) by pre-warming to 37 &#176;C.</li>
	<li>After 1 hr in paraformaldehyde, aspirate off paraformaldehyde and wash gently 3 times with 1x PBS.</li>
	<li>Add 1 ml of pre-warmed TRAP stain into each well of the plate to be stained.</li>
	<li>Place the plate in an incubator at 37 &#176;C for 1 hr, shielded from light.</li>
	<li>After 1 hr, aspirate off the TRAP stain.</li>
	<li>Wash the wells 3 times with pre-warmed deionized water.</li>
	<li>Counterstain the well with Gill&#8217;s Hematoxylin (CAUTION) for 1&#8211;2 min.</li>
	<li>Image osteoclasts using bright field microscopy.</li>
	<li>Wash wells with deionized water until an adequate color intensity of the stain is achieved, typically when nuclei appear blue.</li>
</ol>
8. Osteoclast Resorption Assay
<ol>
	<li>Use a commercially available 24-well polystyrene culture dish that is pre-coated with either bone substrate or an inorganic crystalline calcium phosphate coating.</li>
	<li>Place 2 ml of macrophage stimulating media into each well.</li>
	<li>Add 200,000 freshly isolated bone marrow cells obtained at the end of step 2 to each well.</li>
	<li>Gently agitate the plate prior to placing it in an incubator at 37 &#176;C for 3 days.</li>
	<li>On day 3, change the media to osteoclast differentiation media.</li>
	<li>Perform daily media changes with osteoclast differentiation media for 5&#8211;7 days.</li>
</ol>
9. Quantification of Osteoclast Resorption Activity
<ol>
	<li>After 5&#8211;7 days of in vitro culture in osteoclast differentiation media on a mineralized substrate, analyze the mineralized surface, as described below, to assess the ability of osteoclasts to form resorption pits, an indicator of osteoclastic activity.</li>
	<li>Aspirate media from the resorption assay plate and add 100 &#956;l of 10% bleach solution to each well.</li>
	<li>Incubate the cells with the 10% bleach solution for 15 min at RT.</li>
	<li>Wash the wells 3 times with distilled water.</li>
	<li>Aspirate off any remaining water and shake off excess water from the plate and allow to air dry for 3&#8211;5 hr.</li>
	<li>Counterstain the plate with a commercially available Von Kossa staining kit to allow for easier visualization of the un-resorbed substrate.</li>
	<li>Use bright field microscopy to take images of representative parts of the resorbed surface.</li>
	<li>Using image analysis software, calculate the percentage of the resorbed surface to allow for quantification of osteoclastic resorptive activity.</li>
</ol>

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	<li>Sims, N. A., Martin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+the+activities+of+bone+formation+and+resorption:+a+multitude+of+signals+within+the+basic+multicellular+unit.">Coupling the activities of bone formation and resorption: a multitude of signals within the basic multicellular unit.</a> BoneKEy reports. 3, 481 (2014).</li><li>Kahn, A. J., Simmons, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+cell+lineage+in+bone+using+a+chimaera+of+chick+and+quial+embryonic+tissue.">Investigation of cell lineage in bone using a chimaera of chick and quial embryonic tissue.</a> Nature. 258, 325-327 (1975).</li><li>Walker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+resorption+restored+in+osteopetrotic+mice+by+transplants+of+normal+bone+marrow+and+spleen+cells.">Bone resorption restored in osteopetrotic mice by transplants of normal bone marrow and spleen cells.</a> Science. 190, 784-785 (1975).</li><li>Burger, E. 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=In+vitro+formation+of+osteoclasts+from+long-term+cultures+of+bone+marrow+mononuclear+phagocytes.">In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes.</a> The Journal of experimental medicine. 156, 1604-1614 (1982).</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> The Journal of pathology. 144, 225-226 (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=Bench+to+bedside:+elucidation+of+the+OPG-RANK-RANKL+pathway+and+the+development+of+denosumab.">Bench to bedside: elucidation of the OPG-RANK-RANKL pathway and the development of denosumab.</a> Nature reviews. Drug discovery. 11, 401-419 (2012).</li><li>Brown, J. E., Coleman, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denosumab+in+patients+with+cancer-a+surgical+strike+against+the+osteoclast.">Denosumab in patients with cancer-a surgical strike against the osteoclast.</a> Nature reviews. Clinical oncology. 9, 110-118 (2012).</li><li>Khosla, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+the+OPG/RANKL/RANK+system.">Minireview: the OPG/RANKL/RANK system.</a> Endocrinology. 142, 5050-5055 (2001).</li><li>Boyle, 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=Differential+roles+of+MAPK+kinases+MKK3+and+MKK6+in+osteoclastogenesis+and+bone+loss.">Differential roles of MAPK kinases MKK3 and MKK6 in osteoclastogenesis and bone loss.</a> PloS one. 9, (2014).</li><li>Hofbauer, L. C., Heufelder, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+receptor+activator+of+nuclear+factor-kappaB+ligand+and+osteoprotegerin+in+bone+cell+biology.">Role of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in bone cell biology.</a> Journal of molecular medicine (Berlin, Germany). 79, 243-253 (2001).</li><li>Teramachi, J., 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=Increased+IL-6+Expression+in+Osteoclasts+is+Necessary+but+not+Sufficient+for+the+Development+of+Paget's+Disease+of+Bone.">Increased IL-6 Expression in Osteoclasts is Necessary but not Sufficient for the Development of Paget's Disease of Bone.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. , (2013).</li><li>Catalfamo, 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=Hyperglycemia+induced+and+intrinsic+alterations+in+type+2+diabetes-derived+osteoclast+function.">Hyperglycemia induced and intrinsic alterations in type 2 diabetes-derived osteoclast function.</a> Oral diseases. 19, 303-312 (2013).</li><li>Schueler, J., 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=Intratibial+injection+of+human+multiple+myeloma+cells+in+NOD/SCID+IL-2Rgamma(null)+mice+mimics+human+myeloma+and+serves+as+a+valuable+tool+for+the+development+of+anticancer+strategies.">Intratibial injection of human multiple myeloma cells in NOD/SCID IL-2Rgamma(null) mice mimics human myeloma and serves as a valuable tool for the development of anticancer strategies.</a> PloS one. 8, (2013).</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 in molecular biology (Clifton, N.J). 1130, 307-313 (2014).</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 protocols. , (2008).</li><li>Yamamoto, Y., 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=Osteoblasts+provide+a+suitable+microenvironment+for+the+action+of+receptor+activator+of+nuclear+factor-kappaB+ligand.">Osteoblasts provide a suitable microenvironment for the action of receptor activator of nuclear factor-kappaB ligand.</a> Endocrinology. 147, 3366-3374 (2006).</li><li>Yasuda, 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=Osteoclast+differentiation+factor+is+a+ligand+for+osteoprotegerin/osteoclastogenesis-inhibitory+factor+and+is+identical+to+TRANCE/RANKL.">Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.</a> Proceedings of the National Academy of Sciences of the United States of America. 95, 3597-3602 (1998).</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, 165-176 (1998).</li><li>Teitelbaum, S. L., Ross, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+osteoclast+development+and+function.">Genetic regulation of osteoclast development and function.</a> Nature reviews. Genetics. 4, 638-649 (2003).</li><li>Agas, D., Sabbieti, M. 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J., Udagawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+regulation+of+osteoclast+function.">Hormonal regulation of osteoclast function.</a> Trends in endocrinology and metabolism. 9, 6-12 (1998).</li><li>Nakamura, T., 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=Estrogen+prevents+bone+loss+via+estrogen+receptor+alpha+and+induction+of+Fas+ligand+in+osteoclasts.">Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts.</a> Cell. 130, 811-823 (2007).</li><li>Bellido, T., 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=Regulation+of+interleukin-6,+osteoclastogenesis,+and+bone+mass+by+androgens.+The+role+of+the+androgen+receptor.">Regulation of interleukin-6, osteoclastogenesis, and bone mass by androgens. The role of the androgen receptor.</a> The Journal of clinical investigation. 95, 2886-2895 (1995).</li><li>Roato, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+among+cells+of+bone,+immune+system,+and+solid+tumors+leads+to+bone+metastases.">Interaction among cells of bone, immune system, and solid tumors leads to bone metastases.</a> Clinica., &amp; developmental immunology. 2013, (2013).</li><li>Autio, K. A., Morris, 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=Targeting+bone+physiology+for+the+treatment+of+metastatic+prostate+cancer.">Targeting bone physiology for the treatment of metastatic prostate cancer.</a> Clinical advances in hematolog., &amp; oncology. 11, 134-143 (2013).</li><li>Sottnik, J. L., Keller, E. 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None of the authors has disclosure or conflict of interest to declare.

An ability to easily isolate and cultivate large numbers of osteoclasts in vitro has been responsible for helping to advance understanding of bone biology and osteoclast-mediated diseases. It was the identification of RANKL that lead to this, when it was recently identified as the major regulator of osteoclast formation, differentiation and survival16-18.
It has been our experience that the in vitro cultivation of osteoclasts from bone marrow is largely dependent o...

Bone remodeling is dynamic and involves the coupling of bone formation with bone resorption1. This tightly regulated process is responsible for maintaining the skeleton during normal homeostasis, and in response to injury and disease.
Osteoclasts are unique, multinucleated cells that are capable of resorbing both the organic and inorganic matrices of bone. Osteoclasts are derived from the monocyte/macrophage lineage of the bone marrow2-5. Abnormalities in the function or formation of osteoclasts can result in a variety of clinical pathologies, including common conditions like osteoporosis.
The ability to generate osteoclasts in vitro has allowed for significant advances in our understanding of bone biology6. As a result, new therapeutic agents are emerging to treat osteoclast-related diseases which are responsible for significant morbidities and mortalities7. Homeostatic maintenance of bone mass and strength requires the concerted action of bone-forming osteoblasts and bone-resorbing osteoclasts8,9. Bone homeostasis is altered in a number of diseases, including post-menopausal osteoporosis, in which increased osteoclast activity leads to pathogenic loss of bone mass and density10. With increasing availability of transgenic murine models of human disease, there is more opportunity to decipher the role of the osteoclasts in human bone disease11-13.
Numerous protocols for osteoclast culturing techniques appear in the literature, with many variations described9,12,14. Xing and colleagues describe similar methodology to the protocol described below, in their description of osteoclastogenic assays from murine bone marrow cells. However to release the bone marrow cells following long bone harvest, Xing et al. flush the marrow cavity with &#945;-MEM complete media14. Catalfamo examines the effect of hyperglycemia on osteoclast function and describes a method in which all cells mobilized by bone marrow flushing are cultured for 24 hr, at which point the non-adherent cells are discarded12, a technique also used by Boyle et al.9 These previously published protocols necessitate the practice of flushing the bone marrow, a tedious practice, which also introduces the risk of a needle stick injury and loss of valuable bone marrow, as one must cut both ends of the bone. The protocol, which we describe, implements the use of a mortar and pestle to isolate osteoclasts, which is similar to the method of macrophage isolation described by Weischenfeldt et al.15 
Our experience, however, is that osteoclast isolation and in vitro culture using previously published techniques results in variable outcomes in terms of osteoclast production, often resulting in an inability to cultivate osteoclasts. Therefore, we have devised a protocol that allows for the consistent isolation of mouse bone marrow to produce large numbers of multinucleated osteoclasts in vitro, with an approximate yield of 70-80% of cells initially plated forming macrophages and subsequently osteoclasts, in the presence of osteoclast induction media.

<table><tbody><tr><td>MEM, no glutamine, no phenol red</td><td>Gibco</td><td>51200-038</td><td></td></tr><tr><td>M-CSF, recombinant mouse</td><td>Gibco</td><td>PMC2044</td><td></td></tr><tr><td>Recombinant Mouse TRANCE/RANK L/TNFSF11 (&lt;em&gt;E. coli&lt;/em&gt; expressed)</td><td>R&amp;amp;D Systems</td><td>462-TEC-010</td><td></td></tr><tr><td>Prostaglandin E2</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Histopaque-1077</td><td>Sigma-Aldrich</td><td>10771</td><td></td></tr><tr><td>Acid Phosphatase, Lekocyte (TRAP) kit</td><td>Sigma-Aldrich</td><td>387A</td><td></td></tr><tr><td>Osteoassay bone resorption plates, 24 well plates</td><td>Corning Life Sciences</td><td>3987</td><td></td></tr></tbody></table>

osteoclast derivation from mouse bone marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

The aim of this method was to easily isolate large numbers of osteoclasts in vitro, typically in one week. Successful isolation of large numbers of osteoclasts was confirmed using tartrate-resistant acid phosphatase staining (Figure 1A). Large osteoclasts are visualized as large purple cells with multiple nuclei (typically &#8805; 3 nuclei). Using this protocol, it is common to isolate osteoclasts with as many as 30 nuclei per osteoclast (Figure 1B).
...

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Osteoclast Derivation from Mouse Bone Marrow

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.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

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24.0K Views.  Stanford University School of Medicine. 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.

Video: Osteoclast Derivation from Mouse Bone Marrow

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Cancer Research

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

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.

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Developmental Biology

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.

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 ...

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

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

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.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Immunology and Infection

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Bioengineering

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

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

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

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.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Improved Bone Marrow Injury Model for Intramembranous Bone Repair Studies

Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model

Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage template, the process of intramembranous ossification involves the direct conversion of skeletal stem and progenitor cells (SSPCs) into bone-forming osteoblasts. There are limited surgical methods to model this process in experimental mice. Here, we have improved upon a bone marrow injury model in mice to facilitate the study of bone repair via intramembranous ossification and to assess postnatal regulators of osteogenesis. This method is highly reproducible and user-friendly, and it allows temporal assessment of new bone formation in a short period (3-7 days post-injury) using micro-computed tomography (&#181;CT) and frozen section histology. Furthermore, the contributions of SSPCs and mature osteoblasts can be readily assessed using a combination of fluorescent reporter mice and this intramembranous bone marrow injury model. In clinical contexts, intramembranous bone formation is relevant for healing critical size defects, stabilized fractures, cortical defects, trauma from tumor resections, and joint replacements.

The murine bone marrow injury model is a useful tool for studying postnatal osteogenesis through intramembranous ossification. This simplified surgical protocol describes the bone marrow ablation procedure for downstream assessment of new bone formation and cellular responses following injury.

Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage ...

Osteoclast Derivation from Mouse Bone Marrow

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