JB es apoyado por becas de de Ciencias Naturales e Ingeniería de Investigación (NSERC). Apoyo a este proyecto ha sido proporcionada por los Institutos Canadienses de Investigación en Salud, Grant MOP-67066.

Este protocolo ha sido adaptado de Lutz et al 1. Cosecha y el procesamiento de la médula ósea <ol><li> El aislamiento de la tibia y el fémur: la eutanasia de los donantes del ratón y el spray de las piernas con el 70% de etanol. Sujete el tobillo primera firmemente con unas pinzas romas y empezar a cortar la piel y la musculatura subyacente para exponer la tibia. Para evitar daños en el hueso, corte lento y paralelo a la tibia, dejando intacta la articulación de la rodilla. Para limpiar el fémur, inmovilizar la articulación de la rodilla sujetándolo con unas pinzas romas. Limpie la musculatura con un par de tijeras afiladas y pinzas curvas. Continuar hacia arriba hasta la articulación de la cadera se expone y las tijeras se puede colocar entre la cabeza del fémur y la articulación de la cadera. Quitar los huesos por conjuntos de corte entre el fémur y la cadera y el lugar en tampón fosfato salino (PBS) en el hielo. </li><li> Extracción de médula ósea: Limpiar la musculatura restante de los huesos con tijeras y pinzas curvas. Cortar las epífisis de cada hueso y localizar la cavidad central. Con una jeringa de 10 ml cargada con PBS y una aguja 25G0.5 &quot;, al ras de la médula ósea en un plato recubierto de tejido de Petri. </li><li> Procesamiento de médula ósea: Use la punta de goma del émbolo de una jeringa de 1 ml de disociar la médula ósea a una suspensión de una sola célula (el uso de arriba hacia abajo, no raspar el movimiento). Recoger la médula ósea en un tubo cónico de halcón, y aclarar la médula ósea residual con PBS. </li></ol> Cultivo de células dendríticas <ol><li> Resuspender las células en los medios de comunicación de CC y precursores contar DC en un hemocitómetro (Figura 1). Usted verá las células de diferentes tamaños, los precursores de DC son los más grandes y más brillantes. Diferencialmente contar sólo estas células. A partir de dos fémures y dos tibias de un tipo salvaje C57Bl / 6 ratones (6-8 semanas), usted debe esperar entre 25 a 40 x 10 6 precursores DC total. <img src="/files/ftp_upload/old/769_2008_6_30_3/article769_figure_1.jpg" alt="" width="218" height="217" /> Figura 1 </li><li> Las células de cultivo a una densidad de 2 x 10 5 precursores DC / mL en los medios de comunicación DC complementado con 40 ng / ml recombinante murino GM-CSF. Células de la placa de no tejidos recubiertos con placas de poliestireno de Petri. </li></ol> Añadir los medios de comunicación (día 3) Para actualizar los medios de comunicación, agregar la mitad del volumen total de medio fresco suplementado con 40 ng / ml de GM-CSF. Vuelva a colocar una tercera parte de los medios de comunicación (día 6) y la maduración Para actualizar los medios de comunicación, retire con cuidado la tercera parte del volumen total de los medios de comunicación y sustituir este volumen con nuevos medios de comunicación DC complementado con 40 ng / ml de GM-CSF en el día 6 de la cultura. Si lo desea, las células dendríticas pueden ser estimuladas para la maduración con citoquinas o peaje-como los ligandos del receptor. En el video, los países en desarrollo fueron madurados por la estimulación durante la noche con 5 ng / mL CpG. La cosecha de las células dendríticas DC la cultura es completa (Figura 2). Las células serán tanto en la suspensión y libremente adherido a la placa. Células adheridas se puede quitar raspando el plato con un raspador de cultivo de tejidos y lavar con PBS. El número total de células aumentará 8.5 veces durante el cultivo de una semana y el período de diferenciación, por lo tanto, esperamos cosechar 1-1,6 x 10 6 células / ml. <img src="/files/ftp_upload/old/769_2008_6_30_5/article769_figure_2.jpg" alt="Figura 2" width="407" height="358" /> Figura 2 Reactivos <ol><li> DC los medios de comunicación <ol><li> RPMI-1640 suplementado con: </li><li> 10% de suero fetal bovino </li><li> 100 UI / ml de penicilina </li><li> 100 mg / ml de estreptomicina </li><li> 1% de L-glutamina </li><li> 0,1% 2-mercaptoetanol </li><li> 1X no aminoácidos esenciales </li><li> 1X sodio piruvato </li></ol></li><li> Recombinante murino GM-CSF <ol><li> rmgm-CSF (Peprotech inc, New Jersey, no hay ningún catálogo. 315-03) </li></ol></li></ol>

<ol>
	<li>Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., 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=An+advanced+culture+method+for+generating+large+quantities+of+highly+pure+dendritic+cells+from+mouse+bone+marrow.">An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.</a> J. Immunol. Methods. 223, 77-92 (1999).</li><li>Gilboa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DC-based+cancer+vaccines.">DC-based cancer vaccines.</a> J. Clin. Invest. 117, 1195-1203 (2007).</li></ol>

Los países en desarrollo son útiles para estudios de interacciones inmune innato y adaptativo, y puede ser empleado como un vector de la vacuna. En este video, que han demostrado los pasos para aislar la médula ósea y diferenciar células dendríticas mieloides in vitro. Tras el período de cultivo, estas células se pueden visualizar al microscopio las células tanto como adherentes, que a menudo poseen las dendritas, y las células no adherentes y vuelta. Los países en desarrollo puede ser manipulado para la presentación de antígen...

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culture of myeloid dendritic cells from bone marrow precursors

Células dendríticas mieloides (DC) se utilizan con frecuencia para estudiar las interacciones entre los mecanismos de inmunidad innata y adaptativa y la respuesta temprana a la infección. Debido a que estos son los antígenos más potentes células presentadoras de los países en desarrollo se están utilizando cada vez más como un vector de la vacuna para estudiar la inducción de antígenos específicos de la respuesta inmune. En este vídeo, que muestran el procedimiento para la recolección de la tibia y el fémur de un ratón de los donantes, el procesamiento de la médula ósea y la diferenciación de los países en desarrollo in vitro. Las propiedades de cambio los países en desarrollo después de la estimulación: las células dendríticas inmaduras son fagocitos potentes, mientras que los países en desarrollo maduros son capaces de presentación del antígeno y la interacción con CD4 + y CD8 + T. Este cambio en la actividad funcional se corresponde con la regulación al alza de los marcadores de superficie celular y la producción de citoquinas. Muchos de estos agentes puede ser usado para los países en desarrollo maduro, incluyendo citoquinas y peaje-como los ligandos del receptor. En este vídeo, que muestran las comparaciones de citometría de flujo de la expresión de dos moléculas co-estimulantes, CD86 y CD40, y la citoquina, la IL-12, tras la estimulación durante la noche con el tratamiento con CpG o simuladas. Después de la diferenciación, países en desarrollo puede ser manipulado para su uso como vector de la vacuna o para generar antígenos específicos de la respuesta inmune in vitro de pulso con péptidos o proteínas, o transducidas con vectores virales recombinantes.

Watch this Scientific Journal Video about Culture of myeloid dendritic cells from bone marrow precursors at JoVE.com

Culture of myeloid dendritic cells from bone marrow precursors

Este video muestra el procedimiento para la diferenciación de células dendríticas mieloides de la médula ósea de ratón. El aislamiento de la tibia y el fémur de ratón, y el procesamiento de la médula ósea se demuestran. Imágenes que demuestran la morfología celular, antes y después de la diferenciación, y figuras que representan el fenotipo celular y la IL-12 después de la maduración de la producción con CpG se muestran.

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 ...

culture-of-myeloid-dendritic-cells-from-bone-marrow-precursors

Research

JoVE Journal

Biology

22.4K vistas.  McMaster University. Este video muestra el procedimiento para la diferenciación de células dendríticas mieloides de la médula ósea de ratón. El aislamiento de la tibia y el fémur de ratón, y el procesamiento de la médula ósea se demuestran. Imágenes que demuestran la morfología celular, antes y después de la diferenciación, y figuras que representan el fenotipo celular y la IL-12 después de la maduración de la producción con CpG se muestran.

Video: Culture of myeloid dendritic cells from bone marrow precursors

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Células madre embrionarias humanas: Cultura a partir de células congeladas

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Investigación

Biología

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Cultura de ratón con células madre neurales Precursores

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

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

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% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

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% ...

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 ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

La diferenciación de células madre embrionarias en los precursores de oligodendrocitos

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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 ...

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

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

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.