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.

Tutto il lavoro animale è stato approvato dalla cura degli animali e del Comitato Istituzionale Usa in conformità con le raccomandazioni delineate nella guida per la cura e l&#39;uso di animali da laboratorio del National Institutes of Health. La dissezione 1. arto posteriore delle ossa lunghe <ol><li> Euthanize il mouse in conformità con le linee guida istituzionali. </li><li> Posizionare il mouse in posizione supina e apporre da appuntare tutte e quattro le gambe attraverso le pastiglie del mouse zampa sotto l&#39;articolazione della caviglia. </li><li> Spruzzare il mouse con il 70% di etanolo, accuratamente dousing le gambe. </li><li> Fai una piccola incisione a destra di linea mediana nel basso addome, appena sopra l&#39;anca. </li><li> Estendere l&#39;incisione lungo la gamba e oltre la caviglia. </li><li> Tirare indietro la pelle e tagliare il muscolo quadricipite ancorato all&#39;estremità prossimale del femore per esporre il lato anteriore del femore e il pin out dalla gamba, posizionando il perno con un angolo di 45 gradi dalla scheda. </li><li> Con la fogliae delle forbici contro il lato posteriore del femore, tagliare i femorali dal ginocchio. </li><li> Tirare indietro la pelle ed i muscoli adduttori ancorati alla estremità prossimale del femore per esporre il lato posteriore del femore e il pin dalla gamba, mettendo il perno in un angolo di 45 gradi dalla scheda. </li><li> Con la pinza, tenere l&#39;estremità distale del femore, appena sopra il ginocchio. Guida lame delle forbici su entrambi i lati del femore verso l&#39;anca, facendo attenzione a non tagliare nel femore stesso. </li><li> Dopo aver raggiunto la testa del femore, indicato dalle forbici apertura leggermente, ruotare le forbici con lama superiore delle forbici muovono direttamente sopra la testa femorale di dislocare femore, facendo attenzione a non scattare l&#39;osso sotto della testa femorale. </li><li> Afferrare la parte superiore del femore con la pinza, tagliare il tessuto molle dalla testa del femore per liberarla dal dell&#39;acetabolo. </li><li> Estrarre l&#39;intero osso della gamba, tra cuifemore, ginocchio e tibia, alto e lontano dal corpo, accuratamente tagliando via il tessuto connettivo e muscolare che collega la gamba alla pelle. </li><li> Sovraccaricare l&#39;articolazione della caviglia e di nuovo usare le forbici in un movimento rotatorio di dislocare la tibia. </li><li> Afferrando l&#39;estremità distale della tibia, facendo attenzione a non recidere i tendini, tirare la tibia alto e lontano dal corpo e la lavagna. </li><li> Tagliare qualsiasi tessuto connettivo restante fissaggio del osso lungo al mouse al ginocchio. </li><li> Rimuovere qualsiasi muscolo supplementare o tessuto connettivo attaccata al femore e la tibia. </li><li> Per tutte le applicazioni che richiedono l&#39;osso di mantenere intatte (istologia, istomorfometria, test biomeccanico, ecc.), Procedere con protocolli standard in-house (come nel 4-7). Per isolare il midollo osseo, passare alla sezione 2. </li></ol> 2. Preparazione delle ossa lunghe per isolamento di midollo osseo <ol><li> Utilizzando le pinze, afferrare il femore con la rotula rivolto verso unND l&#39;estremità prossimale (testa del femore) verso il basso. </li><li> Sovraccaricare l&#39;articolazione del ginocchio e usare le forbici in un movimento rotatorio di dislocare la tibia e il femore. </li><li> Tagliare qualsiasi tessuto connettivo che tiene il femore e tibia insieme. </li><li> Utilizzando le pinze, afferrare il femore con il lato anteriore rivolto verso e l&#39;estremità prossimale (lato testa del femore) verso il basso. </li><li> Guida le forbici la diafisi femorale ai condili. </li><li> Delicatamente ruotare le forbici avanti e indietro per rimuovere i condili, rotula, e l&#39;epifisi per esporre metafisi. </li><li> Rimuovere ogni muscolo supplementare o del tessuto connettivo attaccato al femore usando pinze, forbici, e Kimwipes. </li><li> Utilizzando le pinze, afferrare la tibia con il lato anteriore rivolto verso e l&#39;estremità distale (fine caviglia) verso il basso. </li><li> Se l&#39;epifisi tibiale è intatto, guidare le forbici il pozzo tibia ai condili. </li><li> Delicatamente ruotare le forbici avanti e indietro per rimuovere i condili e dell&#39;epifisi per esporre la MetYsis. </li><li> Rimuovere ogni muscolo supplementare o del tessuto connettivo attaccato alla tibia usando pinze, forbici, e Kimwipes. </li></ol> Isolamento 3. midollo osseo <ol><li> Inserire un ago G 18 attraverso il fondo di un tubo di 0,5 ml microcentrifuga. </li><li> Posizionare le ossa lunghe (massimo 2 femori e tibie 2) nel tubo, ginocchio-end verso il basso end verso il basso e chiudere il coperchio. </li><li> Nest il tubo di 0,5 ml microcentrifuga in una provetta da 1,5 ml microcentrifuga. </li><li> Centrifugare le provette annidati a ≥10,000 xg in una microcentrifuga per 15 sec. </li><li> Verificare che il midollo osseo è stata filata su ossa mediante ispezione visiva. Le ossa dovrebbero apparire bianco e ci dovrebbe essere una grande pellet visivo nel tubo più grande. </li><li> Scartare il tubo di 0,5 ml microcentrifuga con le ossa. </li><li> Sospendere il midollo osseo in soluzione appropriata (ad esempio, PBS, terreni di coltura, di buffer FACS) e procedere con il protocollo sperimentale (DNA, RNA, o isolamento delle proteine,analisi FACS, o colture cellulari primarie). </li></ol>

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	<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.

Il protocollo qui descritto è ottimizzato per una rapida dissezione del femore e della tibia del mouse con un minimo di danni al tessuto osseo. Questa tecnica è adatta per un numero di analisi a valle, compresi gli studi biomeccanica, istomorfometria (Figura 1A - B), e l&#39;istologia (Figura 1C) 4,7. Il histomophometric rappresentante ricostruzione micoCT 3D (Figura 1A - B) dimostra che sia l&#39;osso spongioso e corticale guscio sono mantenuti che consente la precisa quantificazione dei parametri strutturali standardizzate per Istomorfometria ossa, compreso il numero trabecolare, lo spessore e la spaziatura; volume osseo; e lo spessore corticale, tra le altre misure 8. La sezione istologica mostra un rappresentante fissati in formalina e decalcificata tibia H &amp; E macchiato (Figura 1C). L&#39;immagine dimostra l&#39;integrità sia del calcificata buno e del midollo osseo cellulari per l&#39;analisi istologica. La procedura di midollo osseo isolamento conserva la sterilità dello spazio midollare, ha bassa movimentazione per ridurre la contaminazione, e non richiede il taglio del osso lungo, riducendo così la perdita di resa midollo osseo. Questo midollo osseo è adatta a molte applicazioni a valle, tra cui citometria a flusso 5 e PCR analisi. Inoltre, questa procedura può essere utilizzata per isolare midollo osseo per colture primarie di cellule di midollo osseo, tra osteoclasti e osteoblasti (Figura 2A - B) 4,6. <img alt="Figura 1" src="/files/ftp_upload/53936/53936fig1.jpg" /> Figura 1. istomorfologiche e analisi istologiche di mouse osso lungo. Ricostruzione microCT tridimensionale di una tibia del mouse mostrando (A) il guscio corticale esterno e (B </s<html> Trong&gt;) dell&#39;osso trabecolare (scala bar = 0,5 mm). (C) istologico H &amp; E macchia di una tibia decalcificata e sezionato (4x). Immagini per gentile concessione di Katherine Weilbaecher, Washington University School of Medicine, Stati Uniti d&#39;America. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53936/53936fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/53936/53936fig2.jpg" /> Figura 2. primaria midollo osseo coltura cellulare per la differenziazione degli osteoclasti e gli osteoblasti. Colorazione (A) trappola per osteoclasti multinucleate dopo 7 giorni in mezzi osteoclastogenico (4x). (B) La fosfatasi alcalina (colore viola) per osteoblasti e alizarin rosso (colore rosso) della macchia per la mineralizzazione dopo 21 giorni di mezzi osteogenico. Immagini per gentile concessione di Katherine Weilbaecher, Washington University School of Medicine, Stati Uniti d&#39;America.iles / ftp_upload / 53936 / 53936fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura.

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.

Murino arto posteriore osso lungo dissezione e isolamento di midollo osseo

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

murine-hind-limb-long-bone-dissection-and-bone-marrow-isolation

Universidad de Cartagena UDEC

Research

JoVE Journal

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.

Isolamento, la cultura e la differenziazione delle cellule stromali del midollo osseo e dei progenitori degli osteoclasti dai topi

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

Ricerca

Biologia dello sviluppo

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).

Isolamento e iniezione endovenosa di Murine derivate dal midollo osseo monociti

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

Immunologia e infezioni

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.

Femorale aspirazione del midollo osseo 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 ...

Medicina

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.

Generazione di un gran numero di progenitori mieloidi e precursori delle cellule dendritiche dal midollo osseo murino utilizzando una romanzo cella ordinamento strategia

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.

Effetto dell'anticorpo Anti-c-fms sulla formazione degli osteoclasti e la proliferazione dei precursori degli osteoclasti 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 ...

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.

Procedure di trapianto di midollo osseo nei topi per studiare l'ematopoiesi clonale

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.

Osteoclasti derivazione dal midollo osseo di topo

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

Biologia

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.

Isolamento dei progenitori dei megacariociti di topo

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://appjove.unicartagenaproxy.elogim.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.