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.

Todos los animales de trabajo fue aprobado por el Cuidado de Animales institucional y el empleo de acuerdo con las recomendaciones descritas en la Guía para el Cuidado y Uso de Animales de Laboratorio de los Institutos Nacionales de Salud. La disección 1. extremidad posterior del hueso largo <ol><li> La eutanasia el ratón de acuerdo con las directrices institucionales. </li><li> Coloque el ratón en una posición supina y colocar fijando las cuatro patas a través de las almohadillas de las patas del ratón debajo de la articulación del tobillo. </li><li> Pulverizar el ratón con un 70% de etanol, rociar a fondo las piernas. </li><li> Hacer una pequeña incisión a la derecha de la línea media en el abdomen inferior, justo por encima de la cadera. </li><li> Extender la incisión en la pierna y más allá de la articulación del tobillo. </li><li> Tire hacia atrás la piel y cortar el músculo cuádriceps anclado al extremo proximal del fémur para exponer la cara anterior del fémur y el pasador hacia fuera de la pierna, colocando el pasador en un ángulo de 45 grados con respecto a la placa. </li><li> Con el blade de la tijera contra el lado posterior del fémur, cortar los tendones de la corva lejos de la articulación de la rodilla. </li><li> Tirar de la piel y los músculos isquiotibiales anclados al extremo proximal del fémur para exponer el lado posterior del fémur y el pasador hacia fuera de la pierna, colocando el pasador en un ángulo de 45 grados con respecto a la placa. </li><li> Con las pinzas, mantenga el extremo distal del fémur, justo por encima de la articulación de la rodilla. Guía de las cuchillas de las tijeras a cada lado del eje femoral hacia la articulación de la cadera, teniendo cuidado de no cortar en la propia fémur. </li><li> Después de alcanzar la cabeza del fémur, indicado por las tijeras se abren ligeramente, torcer la tijera con la hoja superior de la tijera se mueven directamente sobre la cabeza del fémur para dislocar el fémur, con cuidado de no romper el hueso debajo de la cabeza del fémur. </li><li> Agarre la parte superior del eje femoral con el fórceps, cortar el tejido blando lejos de la cabeza femoral para liberarla de acetábulo. </li><li> Tire todo el hueso de la pierna, incluyendofémur, la rodilla, y la tibia, arriba y lejos del cuerpo, cuidadosamente cortar el tejido conectivo y muscular que conecta la pierna a la piel. </li><li> Extender demasiado la articulación del tobillo y otra vez utilizar las tijeras en un movimiento giratorio para dislocar la tibia. </li><li> Agarrando el extremo distal de la tibia, teniendo cuidado de no cortar los tendones, tire de la tibia hacia arriba y lejos del cuerpo y el tablón de anuncios. </li><li> Cortar cualquier tejido conectivo restante fijar el hueso largo para el ratón en la rodilla. </li><li> Eliminar cualquier músculo adicional o tejido conectivo unido al fémur y la tibia. </li><li> Para las aplicaciones que requieren que el hueso se mantienen intactos (histología, la histología, las pruebas biomecánicas, etc.), Proceder con protocolos estándar en la casa (como en el 4-7). Para aislar la médula ósea, pase a la sección 2. </li></ol> 2. Preparación de hueso largo de médula ósea Aislamiento <ol><li> Usando las pinzas, agarre el fémur con la rótula de espaldas a unand el extremo proximal (cabeza femoral) hacia abajo. </li><li> Extender demasiado la articulación de la rodilla y el uso de las tijeras en un movimiento giratorio para dislocar la tibia y el fémur. </li><li> Cortar cualquier tejido conectivo que sostiene el fémur y la tibia juntos. </li><li> El uso de las pinzas, sujete el fémur con el lado anterior de espaldas y el extremo proximal (extremo de la cabeza femoral) hacia abajo. </li><li> Guiar la tijera hasta la diáfisis femoral a los cóndilos. </li><li> gire suavemente las tijeras de ida y vuelta para eliminar los cóndilos, la rótula y la epífisis para exponer la metáfisis. </li><li> Retire cualquier músculo adicional o tejido conectivo unido al fémur usando fórceps, tijeras y Kimwipes. </li><li> El uso de las pinzas, sujete la tibia con el lado anterior de espaldas y el extremo distal (extremo de tobillo) hacia abajo. </li><li> Si la epífisis tibial está intacto, guiar a las tijeras de la diáfisis de la tibia de los cóndilos. </li><li> gire suavemente las tijeras de ida y vuelta para eliminar los cóndilos y la epífisis para exponer el metaphanalysis. </li><li> Retire cualquier músculo adicional o tejido conectivo unido a la tibia utilizando fórceps, tijeras y Kimwipes. </li></ol> Aislamiento 3. médula ósea <ol><li> Empuje una aguja de 18 G a través de la parte inferior de un tubo de 0,5 ml de microcentrífuga. </li><li> Coloque los huesos largos (máximo de 2 fémures y tibias 2) en el tubo, hasta la rodilla extremo hacia abajo termina hacia abajo y cierre la tapa. </li><li> Nido del tubo de microcentrífuga de 0,5 ml en un tubo de microcentrífuga de 1,5 ml. </li><li> Centrifugar los tubos anidados en ≥10,000 xg en una microcentrífuga durante 15 segundos. </li><li> Compruebe que la médula ósea se ha hecho girar hacia fuera de los huesos mediante inspección visual. Los huesos deben aparecen de color blanco y no debe haber una gran pastilla visual en el tubo más grande. </li><li> Desechar el tubo de microcentrífuga de 0,5 ml con los huesos. </li><li> Suspender la médula ósea en solución apropiada (por ejemplo, PBS, medios de cultivo, tampón FACS) y proceder con el protocolo experimental (DNA, RNA, o el aislamiento de proteínas,análisis FACS, o cultivo de células primarias). </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.

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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.

El protocolo descrito aquí está optimizado para una rápida disección del fémur y la tibia de ratón con un mínimo de daños en el tejido óseo. Esta técnica es adecuado para un número de análisis aguas abajo, incluyendo estudios de biomecánica, histomorfometría (Figura 1A - B), y la histología (Figura 1C) de 4,7. El representante histomophometric reconstrucción micoCT 3D (Figura 1A - B) demuestra que tanto el hueso esponjoso y cortical shell se mantienen que permite la cuantificación exacta de los parámetros estructurales normalizados para la histomorfometría de hueso, incluyendo el número trabecular, el grosor, y el espaciamiento; volumen de hueso; y el grosor cortical, entre otras medidas 8. El corte histológico muestra un representante fijado en formalina y descalcificadas tibia H &amp; E manchadas (Figura 1C). La imagen demuestra la integridad tanto de la b calcificadouno y la médula ósea celular para el análisis histológico. El procedimiento de aislamiento de la médula ósea conserva la esterilidad del espacio de la médula ósea, tiene baja manejo para reducir la contaminación, y no requiere el corte del hueso largo, reduciendo así la pérdida de rendimiento de la médula ósea. Esta médula ósea es conveniente para muchas aplicaciones posteriores, incluyendo citometría de flujo 5 y los análisis de PCR. Además, este procedimiento se puede utilizar para aislar de la médula ósea para el cultivo celular primario de células de médula ósea, incluyendo los osteoclastos y los osteoblastos (Figura 2A - B) 4,6. <img alt="Figura 1" src="/files/ftp_upload/53936/53936fig1.jpg" /> Figura 1. histomorfológico y análisis histológicos de ratón hueso largo. MicroCT la reconstrucción tridimensional de una tibia de ratón que muestra (A) la concha cortical externa y (B </s<html> trong&gt;) hueso trabecular (barra de escala = 0,5 mm). (C) histológico tinción con HE de una tibia descalcificada y seccionada (4x). Imágenes cortesía de Katherine Weilbaecher, Facultad de Medicina, Universidad de Washington, EE.UU.. <a href="https://www.jove.com/files/ftp_upload/53936/53936fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53936/53936fig2.jpg" /> Figura 2. Primaria de Médula Ósea cultivo celular para la diferenciación de los osteoclastos y osteoblastos. Tinción (A) para los osteoclastos multinucleadas TRAP después de 7 días en los medios de comunicación osteoclastogénicos (4x). (B) La fosfatasa alcalina (color púrpura) de los osteoblastos y rojo de alizarina (color rojo) para la mineralización de manchas después de 21 días en los medios de comunicación osteogénico. Imágenes cortesía de Katherine Weilbaecher, Facultad de Medicina, Universidad de Washington, EE.UU..iles / ftp_upload / 53936 / 53936fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta 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 Hind Limb largo Bone La disección y el aislamiento de médula ósea

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

Research

JoVE Journal

Immunology and Infection

82.7K vistas.  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

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Ortotópico patas traseras trasplante en ratas

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

Investigación

Inmunología e Infección

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Generación y Etiquetado de murino médula ósea procedentes de células dendríticas con nanocristales Qdot de Estudios de seguimiento

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Investigación de la polarización de macrófagos utilizando la médula ósea macrófagos derivados

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Aislamiento, Purificación y Etiquetado de neutrófilos de médula ósea de ratón de Estudios funcionales y experimentos de transferencia adoptivos

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Médula ósea derivada de macrófagos Producción

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

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).

Aislamiento y la inyección intravenosa de murino médula ósea derivados monocitos

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

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Seguimiento de médula ósea de ratón monocitos<em&gt; En Vivo</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Fémur Window Cámara Modelo de<em&gt; En Vivo</em&gt; Seguimiento de la célula de la médula ósea murina

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Evaluación de los efectos de drogas basados en anticuerpos en médula ósea murina y activación de los macrófagos peritoneales

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.