This work was supported by NCI grant nos. U54CA143803, CA163124, CA093900, and CA143055 to K.J.P. The authors thank the current and past members of the Weilbaecher lab, especially Katherine Weilbaecher, Michelle Hurchla, and Hongju Deng, and members of the Brady Urological Institute, especially members of the Pienta laboratory for critical reading of the manuscript.

All animal work was approved by the Institutional Animal Care and Use Committee in accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
1. Hind Limb Long Bone Dissection
<ol>
	<li>Euthanize the mouse in accordance with institutional guidelines.</li>
	<li>Position the mouse in a supine position and affix by pinning all four legs through the mouse paw pads below the ankle joint.</li>
	<li>Spray the mouse with 70% ethanol, thoroughly dousing the legs.</li>
	<li>Make a small incision to the right of midline in the lower abdomen, just above the hip.</li>
	<li>Extend the incision down the leg and past the ankle joint.</li>
	<li>Pull back the skin and cut the quadriceps muscle anchored to proximal end of the femur to expose the anterior side of the femur and pin out from the leg, placing the pin at a 45-degree angle from the board.</li>
	<li>With the blade of the scissors against the posterior side of the femur, cut the hamstrings away from the knee joint.</li>
	<li>Pull back the skin and the hamstring muscles anchored to proximal end of the femur to expose the posterior side of the femur and pin out from the leg, placing the pin at a 45-degree angle from the board.</li>
	<li>With the forceps, hold the distal end of the femur, just above the knee joint. Guide the blades of the scissors on either side of the femoral shaft towards the hip joint, being careful not to cut into the femur itself.</li>
	<li>After reaching the femoral head, indicated by the scissors opening slightly, twist the scissors with the top blade of the scissors moving directly over the femoral head to dislocate the femur, being careful not to snap the bone below the femoral head.</li>
	<li>Grasp the top of the femoral shaft with the forceps, cut the soft tissue away from the femoral head to release it from the acetabulum.</li>
	<li>Pull the entire leg bone, including femur, knee, and tibia, up and away from the body, carefully cutting away the connective tissue and muscle connecting the leg to the skin.</li>
	<li>Overextend the ankle joint and again use the scissors in a twisting motion to dislocate the tibia.</li>
	<li>Grasping the distal end of the tibia, taking care not to sever the tendons, pull the tibia up and away from the body and the pin board.</li>
	<li>Cut any remaining connective tissue attaching the long bone to the mouse at the knee.</li>
	<li>Remove any additional muscle or connective tissue attached to the femur and the tibia.</li>
	<li>For any applications that require the bone to remain intact (histology, histomorphometry, biomechanical testing, etc.), proceed with standard in-house protocols (as in 4-7). To isolate bone marrow, proceed to section 2.</li>
</ol>
2. Long Bone Preparation for Bone Marrow Isolation
<ol>
	<li>Using the forceps, grasp the femur with the patella facing away and the proximal end (femoral head) down.</li>
	<li>Overextend the knee joint and use the scissors in a twisting motion to dislocate the tibia and femur.</li>
	<li>Cut any connective tissue holding the femur and tibia together.</li>
	<li>Using the forceps, grasp the femur with the anterior side facing away and the proximal end (femoral head end) down.</li>
	<li>Guide the scissors up the femoral shaft to the condyles.</li>
	<li>Gently rotate the scissors back and forth to remove the condyles, the patella, and the epiphysis to expose the metaphysis.</li>
	<li>Remove any additional muscle or connective tissue attached to the femur using forceps, scissors, and Kimwipes.</li>
	<li>Using the forceps, grasp the tibia with the anterior side facing away and the distal end (ankle end) down.</li>
	<li>If the tibial epiphysis is intact, guide the scissors up the tibia shaft to the condyles.</li>
	<li>Gently rotate the scissors back and forth to remove the condyles and epiphysis to expose the metaphysis.</li>
	<li>Remove any additional muscle or connective tissue attached to the tibia using forceps, scissors, and Kimwipes.</li>
</ol>
3. Bone Marrow Isolation
<ol>
	<li>Push an 18 G needle through the bottom of a 0.5 ml&#160;microcentrifuge tube.</li>
	<li>Place the long bones (maximum of 2 femurs and 2 tibiae) into the tube, knee-end down end down and close the lid.</li>
	<li>Nest the 0.5 ml microcentrifuge tube in a 1.5 ml microcentrifuge tube.</li>
	<li>Centrifuge the nested tubes at &#8805;10,000 x g in a microcentrifuge for 15 sec.</li>
	<li>Verify that the bone marrow has been spun out of the bones by visual inspection. The bones should appear white and there should be a large visual pellet in the larger tube.</li>
	<li>Discard the 0.5 ml microcentrifuge tube with the bones.</li>
	<li>Suspend the bone marrow in appropriate solution (e.g., PBS, culture media, FACS buffer) and proceed with experimental protocol (DNA, RNA, or protein isolation, FACS analysis, or primary cell culture).</li>
</ol>

<ol>
	<li>McHayleh, W. M., Ellerman, J., Roodman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+80.">Ch. 80.</a> Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. , 379-381 (2008).</li><li>Weilbaecher, K. N., Guise, T. A., McCauley, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+to+bone:+a+fatal+attraction.">Cancer to bone: a fatal attraction.</a> Nat Rev Cancer. 11 (6), 411-425 (2011).</li><li>Pedersen, E. A., Shiozawa, Y., Pienta, K. J., Taichman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prostate+cancer+bone+marrow+niche:+more+than+just+'fertile+soil.">The prostate cancer bone marrow niche: more than just 'fertile soil.</a> Asian J Androl. 14 (3), 423-427 (2012).</li><li>Amend, S. 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=Thrombospondin-1+regulates+bone+homeostasis+through+effects+on+bone+matrix+integrity+and+nitric+oxide+signaling+in+osteoclasts.">Thrombospondin-1 regulates bone homeostasis through effects on bone matrix integrity and nitric oxide signaling in osteoclasts.</a> J Bone Miner Res. 30 (1), 106-115 (2015).</li><li>Hurchla, M. A., 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=The+epoxyketone-based+proteasome+inhibitors+carfilzomib+and+orally+bioavailable+oprozomib+have+anti-resorptive+and+bone-anabolic+activity+in+addition+to+anti-myeloma+effects.">The epoxyketone-based proteasome inhibitors carfilzomib and orally bioavailable oprozomib have anti-resorptive and bone-anabolic activity in addition to anti-myeloma effects.</a> Leukemia. 27 (2), 430-440 (2013).</li><li>Rauch, D. A., 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=The+ARF+tumor+suppressor+regulates+bone+remodeling+and+osteosarcoma+development+in+mice.">The ARF tumor suppressor regulates bone remodeling and osteosarcoma development in mice.</a> PLoS One. 5 (12), e15755 (2010).</li><li>Su, X., 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=The+ADP+receptor+P2RY12+regulates+osteoclast+function+and+pathologic+bone+remodeling.">The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling.</a> J Clin Invest. 122 (10), 3579-3592 (2012).</li><li>Dempster, D. W., 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=Standardized+nomenclature,+symbols,+and+units+for+bone+histomorphometry:+a+2012+update+of+the+report+of+the+ASBMR+Histomorphometry+Nomenclature+Committee.">Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee.</a> J Bone Miner Res. 28 (1), 2-17 (2013).</li><li>Begley, C. G., Ellis, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+development:+Raise+standards+for+preclinical+cancer+research.">Drug development: Raise standards for preclinical cancer research.</a> Nature. 483 (7391), 531-533 (2012).</li><li>Festing, M. F., Altman, 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=Guidelines+for+the+design+and+statistical+analysis+of+experiments+using+laboratory+animals.">Guidelines for the design and statistical analysis of experiments using laboratory animals.</a> ILAR J. 43 (4), 244-258 (2002).</li></ol>

The authors have nothing to disclose.

We present a simple and efficient method for removal of mouse hind long bones and subsequent bone marrow isolation. This method maintains the high structural and cellular integrity of the bones and bone marrow and has low handling time, minimizing the likelihood of user-induced fracture or bone scoring that may influence downstream analyses. In addition, the centrifugation method for isolating bone marrow does not require cutting the bone to expose the bone marrow space or fluid to flush the bone marrow, reducing potential points of contamination. Moreover, the centrifuge technique is relatively high-throughput with lower hands-on time than other methods, thus reducing processing time.
High variation is inherent to in vivo mouse studies due to high mouse-to-mouse phenotypic variation. In order to maximize the research impact of expensive and labor-intensive mouse studies, it is critical to minimize technical experimental error9,10. Time from animal sacrifice to downstream analysis or tissue fixation introduces experimental variation that may overcome subtle changes and reduce large differences between groups. Therefore, rapid processing of samples is essential for accurate data analysis. The long bone dissection and bone marrow isolation techniques described here are optimized for rapid processing of animals and samples to reduce technical variation.
This protocol can be widely applied to many research fields, including investigation of the bone tissue itself or interrogation of the cells of the bone marrow. In addition, this straightforward approach to long bone dissection will enable researchers in related fields to directly interrogate bone contributions in order to expand our knowledge of bone marrow dysfunction in otherwise understudied pathologies.

The study of long bones and the cells of the bone marrow is central to a myriad of research disciplines, including, but not limited to, bone biology, cancer biology, immunology, hematology, and biomechanics. The bone is a highly dynamic organ that together with the cartilage forms the skeleton to provide mechanical support against loading and protection of the internal organs. In addition, the mineral components of bone are a storage sink for the critical signaling molecules calcium and phosphorus, as well as other factors1. Finally, bones house the bone marrow and, together with metabolically active bone forming osteoblasts and bone resorbing osteoclasts, provide the stem cell niche necessary for the maintenance of hematopoietic and lymphoid cell populations.
Bone and bone marrow are affected in many disorders, often leading to bone marrow dysfunction, severe bone pain, and pathologic fracture. Bone is a common site of metastasis in many solid tumors, most notably breast cancer and prostate cancer, where tumor cells directly engage the bone marrow niche to initiate the vicious cycle of bone metastasis and displace hematopoietic stem cells2,3. Hematopoietic malignancies including myeloma and leukemia are characterized by bone marrow dysfunction as well as deregulation of healthy bone remodeling1. Other non-malignant skeletal disorders are also active areas of research, such as osteoarthritis, osteoporosis, scoliosis, and rickets. Even in an otherwise healthy individual, biomechanical failure in a bone leads to a painful fracture. All of these disorders represent active areas of research with the goal of identifying new preventative measures and treatment regimens to reduce morbidity and mortality.
To research the plethora of roles of the bone and the bone marrow, both under physiologic and pathologic conditions, it is critical for researchers to have a simple and efficient standardized method for dissection of the mouse long bones for rapid processing of large in vivo experiments. The dissection protocol outlined here is suitable for all long bone analyses including ex vivo imaging, histology, histomorphometry, and strength testing, among others. Similarly, a standardized bone marrow isolation method with high bone marrow cell recovery and low inter-user variability is important for experimental analysis such as fluorescence-activated cell sorting (FACS) or quantitative PCR (qPCR) as well as downstream applications such as primary cell culture of bone marrow cells.

<table><tbody><tr><td>Pinboard</td><td></td><td></td><td></td></tr><tr><td>Pins</td><td></td><td></td><td></td></tr><tr><td>70% Ethanol</td><td></td><td></td><td></td></tr><tr><td>Dissection scissors&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Dissection forceps</td><td></td><td></td><td></td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark</td><td>34120</td><td></td></tr><tr><td>16 G needle</td><td></td><td></td><td></td></tr><tr><td>1.5 ml Microcentrifuge tube</td><td></td><td></td><td></td></tr><tr><td>0.5 ml Microcentrifuge tube</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td></td></tr></tbody></table>

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.

The protocol described here is optimized for rapid dissection of the mouse femur and tibia with a minimum of damage to the bone tissue. This technique is suitable for a number of downstream analyses, including biomechanics studies, histomorphometry (Figure 1A - B), and histology (Figure 1C)4,7. The representative histomophometric micoCT 3D reconstruction (Figure 1A - B) demonstrates that both the cancellous bone and cortical shell are maintained that allows for accurate quantitation of the standardized structural parameters for bone histomorphometry, including trabecular number, thickness, and spacing;&#160;bone volume;&#160;and cortical thickness, among other measures8. The representative histologic section shows an H&#38;E stained formalin-fixed and decalcified tibia (Figure 1C). The image demonstrates the integrity of both the calcified bone and cellular bone marrow for histologic analysis.
The bone marrow isolation procedure preserves the sterility of the bone marrow space, has low handling to reduce contamination, and does not require cutting of the long bone, thus reducing loss of bone marrow yield. This bone marrow is suitable for many downstream applications, including flow cytometry5 and PCR analyses. In addition, this procedure can be used to isolate bone marrow for primary cell culture of bone marrow cells, including osteoclasts and osteoblasts (Figure 2A - B)4,6.
<img alt="Figure 1" src="/files/ftp_upload/53936/53936fig1.jpg" /> 
Figure 1. Histomorphological and Histological Analyses of Mouse Long Bone. Three-dimensional microCT reconstruction of a mouse tibia showing (A) the outer cortical shell and (B) trabecular bone (scale bar = 0.5 mm). (C) Histological H&#38;E stain of a decalcified and sectioned tibia (4x). Images courtesy of Katherine Weilbaecher, Washington University School of Medicine, USA. <a href="https://www.jove.com/files/ftp_upload/53936/53936fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53936/53936fig2.jpg" /> 
Figure 2. Primary Bone Marrow Cell Culture for Differentiation of Osteoclasts and Osteoblasts. (A) TRAP staining for multinucleated osteoclasts after 7 days in osteoclastogenic media (4x). (B) Alkaline phosphatase (purple color) for osteoblasts and alizarin red (red color) stain for mineralization after 21 days in osteogenic media. Images courtesy of Katherine Weilbaecher, Washington University School of Medicine, USA. <a href="https://www.jove.com/files/ftp_upload/53936/53936fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation at JoVE.com

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

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, ...

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Research

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Immunology and Infection

82.9K Views.  Johns Hopkins University. 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.

Video: Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

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 ...

Developmental Biology

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Medicine

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

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

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by osteoclasts in response to different stimuli. Osteoclast precursors differentiate into multinuclear osteoclasts in response to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL). Under pathologic conditions, the cytokine profile is different and involves a mixture of inflammatory cytokines. Tumor necrosis factor alpha (TNF-&#945;) is one of the most important cytokines as it is found in large amounts in areas involved with inflammatory osteolysis. The purpose of this protocol is to provide a method by which murine bone marrow is isolated to generate osteoclasts through induction with M-CSF and either RANKL or TNF-&#945; which will be subsequently inhibited by increasing doses of anti-c-fms antibody, the receptor for M-CSF. This experiment highlights the therapeutic value of anti-c-fms antibody in diseases of inflammatory bone resorption.

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-&#945;) and their arrest by anti-c-fms antibody, M-CSF receptor.

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

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.

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 ...

Biology

Isolation of Mouse Megakaryocyte Progenitors

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through ...

Utilizing Low Oxygen Tension to Reduce Hematopoietic Cells in Murine Bone Marrow Stromal Cell Cultures

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this reason, BMSCs, which support hematopoiesis and contribute to all the functions of the skeleton, continue to be widely used to study multipotent mesenchymal progenitors (MMPs) and to infer SSC function. Moreover, given the breadth of transgenic murine models used to study musculoskeletal diseases, the use of BMSCs also serves as a powerful tool to examine the molecular mechanisms regulating MMPs and SSCs. However, common isolation procedures for murine BMSCs result in over 50% of recovered cells being of hematopoietic origins, potentially hindering the interpretation of the data generated during these studies. Here, we describe a method using low oxygen tension or hypoxia for the selective elimination of CD45+ cells in BMSC cultures. Importantly, this method can be easily implemented to not only reduce hemopoietic contaminants but to also enhance the percentage of MMPs and putative SSCs in BMSC cultures.

A routine culture of bone marrow stromal cells (BMSCs) leads to the isolation of heterogeneous cell populations, with many cells being of hematopoietic origins. Here, we describe a method that utilizes low oxygen tension to greatly reduce hematopoietic contaminants in murine BMSC cultures.

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this ...

Isolation and Culture of Bone Marrow Stromal Cells from a Mouse Tibia

Source: Maridas, D. E., et al. <a href="https://app.jove.com/v/56750">Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.</a> J. Vis. Exp.(2018)
In this video, we demonstrate the isolation and culture of bone marrow stromal cells from the long bones of mice. The isolated adherent BMSCs can be used for further analysis.