Institutos Nacionales de Salud Kirchstein beca beca predoctoral AI-64128 (MPM), GM-41.514 (MDC), GM-48071 (IP)

1) Retire los dos huesos de fémur de un ratón <ol><li> Use las tijeras para cortar la disección muscular y exponer el hueso del fémur por encima y por debajo de las articulaciones (rodilla y cadera). Sujete el centro del fémur con unas pinzas de disección y corte por encima y por debajo de las articulaciones de dejar la mayor cantidad de la epífisis intacto como sea posible. </li><li> Limpie tanto músculo como sea posible con unas tijeras pequeñas disección. Transferencia del fémur en un plato de RPMI. </li><li> Todos los procedimientos deben llevarse a cabo en el barrio a partir de ahora, sólo con medio estéril, instrumentos, puntas de las pipetas y placas de cultivo. </li></ol> 2) Esterilizar los huesos del fémur: <ol><li> En la capilla, la transferencia de los huesos de la RPMI a una placa de cultivo de pequeña llena de etanol al 70% en el hielo. </li><li> Permita que los huesos en remojo en etanol durante 5 minutos en hielo. </li></ol> 3) Recoger la médula ósea bajo condiciones estériles: <ol><li> La transferencia de los huesos a un plato de cultivo que contienen pequeñas filtrado estéril de medios Primaria cultura DC. </li><li> Con pinzas estériles, sostener el hueso en un plato de descartar o tubo, y con unas tijeras estériles, cortar cada epiphesis (extremos de los huesos) fuera. El corte se debe exponer la médula ósea, que es de color rojo brillante en el centro del hueso. </li><li> Con una jeringa (aguja de calibre 26-28), absorben alrededor de 100-200 ml de medio estéril. </li><li> Mantener el hueso con la pinza estéril sobre un plato fresco de medio estéril e inserte la aguja en un lado del hueso. La posición de la punta de la aguja en la parte superior del hueso y poco a poco lavar el hueso con los medios de comunicación estéril. Si usted está en el centro del hueso, la aguja debe deslizarse fácilmente. </li><li> La médula ósea hace desaparecer, ya sea en pequeños trozos o en una sola pieza. Debe ser purgada de los huesos y en el plato de los medios de comunicación estéril. </li><li> Repita este paso según sea necesario lavar completamente el tuétano de los huesos. </li><li> Cuando el hueso se limpia, será blanco y translúcido. </li><li> Repita este procedimiento con el hueso del fémur otros. Desechar cada hueso del fémur, una vez que esté limpio. </li></ol> 4) Vuelva a suspender y girar las células de la médula ósea: <ol><li> La transferencia de células a un tubo estéril halcón. </li><li> Si la médula ósea está intacta, con mucho cuidado la pipeta los medios de comunicación de arriba a abajo en el plato, para romper la médula en una suspensión de células individuales. Esto puede tardar varios minutos y se debe hacer lentamente para evitar la muerte de las células por la fuerza. </li><li> Centrifugar las células como lo haría con cualquier otra células de mamíferos. </li></ol> 5) lisis celular: <ol><li> Retire el tubo de la centrífuga después de la vuelta se hace y se vierte fuera de los medios de comunicación (decantación) en una pérdida pequeña (50 ml tubo Falcon) en la campana. </li><li> Resuspender pellet suavemente agitando el fondo del tubo. </li><li> El uso de agua estéril, filtrada y DPBS 10x o 10x PBS, llevar a cabo una lisis de agua para eliminar los glóbulos rojos (GR) con los siguientes volúmenes: <ul><li> 900 ml de agua estéril, filtrada </li><li> 100 ml de DPBS 10x o 10x PBS </li></ul></li><li> Añadir 100 ml de PBS 10x segundo 50-10 después de añadir el agua. Es muy importante hacer esto de inmediato para que sólo los glóbulos rojos son lisadas. Es una buena idea tener dos pipetas de lleno y listo. </li><li> Añadir 5 ml - 10 ml de agua estéril DC medios de comunicación básicas. </li></ol> 6) contar las células: <ol><li> En la capilla, la mezcla de 6 a 11 ml de células de médula ósea girando suavemente el tubo con una inversión pequeña, una inclinación de 45 grados (no para que los medios de comunicación toca la parte superior del tubo) </li><li> Quitar 10 ml de los medios de comunicación en un tubo estéril para contar. </li><li> Vuelva a tapar las células y se centrifuga de nuevo. </li></ol> 7) Las células dendríticas Revestimiento: <ol><li> Las células deben ser plateado con una densidad de 1 x 106/ml. </li><li> Eliminar las células de la centrífuga, los medios de decantación y las células de una nueva suspensión en la cantidad adecuada de Primaria los medios de comunicación de CC para la siembra. </li><li> Lugar en el cultivo de tejidos incubadora. </li></ol> 8) Cuidado de la Cultura y de maduración: Siempre revise los cultivos antes de añadir más medios de comunicación o utilizar en cualquier experimento. Comprobar el estado general de salud de las células y buscar una posible contaminación. DÍA 0: Las células dendríticas plateado. DIA 3: Retire el 75% de los medios de comunicación y las células no adherant y añadir de nuevo los medios de comunicación de CC primaria. DIA 6: después de la siembra inicial de la célula: la placa de Re-las células mediante el siguiente procedimiento: <ol><li> Eliminar los medios de comunicación, que debe contener algunos países en desarrollo no adherant en este punto, y colocar en un tubo estéril de 50 ml halcón, dejando las placas ligeramente húmeda (no quiere las células adherentes se seque) </li><li> Añadir 3 mM EDTA en PBS a cada placa y dejar reposar durante 5 minutos. </li><li> Mantener la placa en un ángulo de 45 grados y suavemente pipeta los medios de comunicación hacia arriba y abajo contra la parte inferior de la placa suavemente para desalojar no adherant células. </li><li> EDTA / PBS debe ser más gruesa que indica las células son recogidas de la parte inferior de laplato. </li><li> Después de varios minutos de esto, mezcla de células se pueden transferir al tubo Falcon de 50 ml. </li><li> Girar todas las células después de realizar este procedimiento para cada plato. </li><li> Conde y la placa de las células a una densidad de 1 x 10 ^ 6 por placa en la secundaria los medios de comunicación de CC en los nuevos platos de 10 cm estéril cultura. Volver a las células de cultivo de tejidos incubadora. </li></ol> DÍA 11.10 Los países en desarrollo maduro listo para la carga de estimulación / antígeno. 9) la estimulación de células dendríticas: <ol><li> Use LPS (lipopolyscaccharide) en combinación con el péptido deseado para la activación y la carga de péptidos. Dependiendo de su fuente de LPS, diferentes condiciones de estimulación puede dar mejores resultados. Además, la cantidad deseada de péptido para la presentación de DC debe ser optimizado. Condiciones de estimulación en este protocolo fueron de 100 ng / mL de LPS y 100 ug / mL OVA (ovoalbúmina). </li><li> Antígeno de carga / tratamiento con LPS durante 18-24 horas antes de la cosecha / uso. </li></ol> Los medios de comunicación las células dendríticas: filtro estéril después de todo ha sido añadido Por favor, consulte la sección de discusión para la revisión de las diferentes condiciones de células dendríticas y la cultura para el fenotipo de células dendríticas resultante. Estas condiciones de cultivo están diseñadas para producir células dendríticas maduras que rápidamente se casa a los ganglios linfáticos de drenaje, 18-24 horas después de la inyección subcutánea y antígeno presente de manera eficiente. Primaria DC medios de comunicación: filtro estéril después de todo lo que se ha añadido <ul><li> 500 ml IMDM (quitar 55-60 ml de volumen final de 500 ml) </li><li> 50 ml de FBS inactivado por calor </li><li> 5 ml de 200 mM L-Gln, la concentración final de 2 mM </li><li> 100 UI / ml de penicilina y 100 mg / ml de estreptomicina (5 ml de 10.000 UI / ml Pen y 10000 mg / ml de caldo de estreptococo) </li><li> 50 uM B-Me (14,3 M B-Me acciones, diluir 1:100 en campana química, utilizar 35 ul por cada 100 ml de medio por 50 la concentración micromolar) </li><li> 20-30 ng / ml de GM-CSF </li><li> 100-400 UI / mL de IL-4 o menos 10 a 40 ng / mL </li></ul> Los medios de comunicación SecondaryDC para la maduración * 100 ng / ml de TNF-alfa (añadir a los medios de comunicación primaria DC) * Nota: GM-CSF + IL-4 debe producir células dendríticas inmaduras. El uso de GM-CSF + IL-4 (400 IU / ul) + TNF-alfa (100 ug / ml), se crean células que se asemejan a madurar células dendríticas derivadas de monocitos. Las células dendríticas maduras se sabe que no ocupan antígeno tan eficientemente como células dendríticas inmaduras y las culturas pueden tener endógeno TNF-a liberados de las células del estroma 4.

<ol>
	<li>Miller, M. J., Safrina, O., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+single+cell+dynamics+of+CD4++T+cell+activation+by+dendritic+cells+in+lymph+nodes.">Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes.</a> J Exp Med. 200, 847-856 (2004).</li><li>Henrickson, S. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+functions+of+IL-4+receptor+types+I+and+II+for+dendritic+cell+maturation+and+IL-12+production+and+their+dependency+on+GM-CSF.">Differential functions of IL-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSF.</a> J Immunol. 169, 3574-3580 (2002).</li><li>Xu, Y., Zhan, Y., Lew, A. M., Naik, S. H., Kershaw, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+development+of+murine+dendritic+cells+by+GM-CSF+versus+Flt3+ligand+has+implications+for+inflammation+and+trafficking.">Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking.</a> J Immunol. 179, 7577-7584 (2007).</li><li>Inaba, K., Swiggard, W. J., Steinman, R. M., Romani, N., Schuler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+dendritic+cells.">Isolation of dendritic cells.</a> Curr Protoc Immunol. 3, (2001).</li><li>Winzler, C., 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=Maturation+stages+of+mouse+dendritic+cells+in+growth+factor-dependent+long-term+cultures.">Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures.</a> J Exp Med. 185, 317-328 (1997).</li><li>Koch, F., 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=Tumor+necrosis+factor+alpha+maintains+the+viability+of+murine+epidermal+Langerhans+cells+in+culture,+but+in+contrast+to+granulocyte/macrophage+colony-stimulating+factor,+without+inducing+their+functional+maturation.">Tumor necrosis factor alpha maintains the viability of murine epidermal Langerhans cells in culture, but in contrast to granulocyte/macrophage colony-stimulating factor, without inducing their functional maturation.</a> J Exp Med. 171, 159-171 (1990).</li><li>Miller, M. J., Hejazi, A. S., Wei, S. H., Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+repertoire+scanning+is+promoted+by+dynamic+dendritic+cell+behavior+and+random+T+cell+motility+in+the+lymph+node.">T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node.</a> Proc Natl Acad Sci U S A. 101, 998-1003 (2004).</li><li>Wei, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++signals+in+CD4++T+cells+during+early+contacts+with+antigen-bearing+dendritic+cells+in+lymph+node.">Ca2+ signals in CD4+ T cells during early contacts with antigen-bearing dendritic cells in lymph node.</a> J Immunol. 179, 1586-1594 (2007).</li><li>Castellino, F., 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=Chemokines+enhance+immunity+by+guiding+naive+CD8++T+cells+to+sites+of+CD4++T+cell-dendritic+cell+interaction.">Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction.</a> Nature. 440, 890-895 (2006).</li></ol>

Las células dendríticas son los principales mediadores de la respuesta inmune adaptativa y el antígeno de células más eficientes presentar caracterizado hasta la fecha. Métodos para el consumo humano y la cultura del ratón de células dendríticas en la literatura difieren en el tipo de citoquinas para influir en el desarrollo de diferentes tipos de células dendríticas. En particular, el GM-CSF, Flt3L, IL4, IL13, TNF-alfa e IFN-gamma se utilizan en diferentes combinaciones para producir maduro, inmaduro, inflamatoria y de estado es...

generation of bone marrow derived murine dendritic cells for use in 2-photon imaging

Existen varios métodos para la preparación de las células dendríticas murinas se puede encontrar en la literatura. A continuación, presentamos un método que produce más del 85% CD11c células dendríticas de alta en la cultura de que el hogar de los ganglios linfáticos que drenan después de la inyección subcutánea, y presentan el antígeno a las células T antígeno específicas (ver video). Además, utilizamos instrumentos Essen Incucyte para realizar un seguimiento de maduración de células dendríticas, donde, el día 10, la morfología de las células en cultivo es típico de una célula madura dendríticas y &lt;85% de las células son CD11chigh. El estudio de la presentación de antígenos en los ganglios linfáticos periféricos de dos fotones de imagen reveló que hay tres fases distintas de las células dendríticas y la interacción de las células T 1, 2. La Fase I se compone de breves contactos en serie entre las células T antígeno específicas muy móviles y el antígeno de llevar a las células dendríticas 1, 2. La segunda fase está marcada por los contactos prolongados entre los antígenos específicos de células T y células dendríticas antígeno teniendo 1, 2. Por último, la fase III se caracteriza por células T desprenderse de las células dendríticas, la recuperación de la motilidad y comienza a dividir 1, 2. Este es un ejemplo del tipo de antígeno específico de las interacciones que pueden ser analizados por dos fotones de imágenes de antígenos de células cargadas de seguimiento marcado tinte células dendríticas.

Watch this Scientific Journal Video about Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging at JoVE.com

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

La presentación de antígenos en los órganos linfoides secundarios por las células dendríticas es fundamental para el inicio de la célula T mediada por la respuesta inmune adaptativa. Aquí se demuestra la cultura de la médula ósea procedentes de células dendríticas murinas, la activación, y el etiquetado de dos fotones de imágenes.

Generación de médula ósea procedentes de células murinas dendríticas para su uso en dos fotones de imágenes

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

generation-bone-marrow-derived-murine-dendritic-cells-for-use-2

Research

JoVE Journal

Biology

31.0K vistas.  University of California, Irvine (UCI). La presentación de antígenos en los órganos linfoides secundarios por las células dendríticas es fundamental para el inicio de la célula T mediada por la respuesta inmune adaptativa. Aquí se demuestra la cultura de la médula ósea procedentes de células dendríticas murinas, la activación, y el etiquetado de dos fotones de imágenes.

Video: Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

La disección y 2-Photon imagen de los ganglios linfáticos periféricos en ratones

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

Investigación

Biología

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

En imágenes in situ del timo de ratón utilizando 2-Photon Microscopía

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

Un método para el 2-Photon imágenes del flujo sanguíneo en la neocorteza a través de una ventana craneal

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

En Vivo 2-Photon imágenes de calcio en la capa 2 / 3 de los ratones

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Cultura de células dendríticas mieloides a partir de precursores de médula ósea

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Mensajeras de las células hematopoyéticas de la médula ósea

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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.

Osteoclastos Derivación de médula ósea de ratón

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

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Expresión de proteínas fluorescentes de fusión en murinas médula ósea células dendríticas y macrófagos

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...