La recolección de médula ósea de fémures y tibias <ol><li> El primer paso del procedimiento consiste en la cosecha de médula ósea de los fémures y tibias de un 6 - a la del ratón de 12 semanas de edad (estamos utilizando ratones Balb / c, pero que puede hacer este procedimiento con cualquier cepa de ratón). Para empezar, la eutanasia el ratón por inhalación de CO 2 (no se mostrará). Fémur y la tibia de la cosecha de las patas traseras de cada uno. Luego, con una cuchilla de afeitar, cortar los extremos del fémur para eliminar la cadera y la rodilla y para exponer la médula ósea. </li><li> De manera similar, cortar los extremos de la tibia para eliminar la rodilla y el tobillo y las regiones adyacentes para exponer la médula ósea. </li><li> Tome una jeringa de 3 ml con una aguja de calibre 26 y lo rellenamos con RPMI suplementado con FBS al 10%, penicilina, estreptomicina, y beta-mercaptoetanol (BME = 100 um). Con unas pinzas, sostener el hueso en una placa de Petri que contiene RPMI medios de comunicación. Inserte la aguja de la jeringa en un extremo del hueso y el émbolo para eliminar la médula ósea. La aguja de la jeringa se puede mover arriba y abajo dentro del hueso para eliminar la médula ósea residual. </li><li> Repita el procedimiento con todos los huesos, y luego proceder con el establecimiento de los cultivos. </li></ol> El establecimiento de las Culturas <ol><li> Para establecer los cultivos de médula ósea, una pipeta de la médula ósea lavado de arriba a abajo varias veces para romper el tejido en una suspensión de células. </li><li> Colocar un filtro de 70 micras en la parte superior de un tubo cónico de 50 ml, y añadir la suspensión celular en el filtro. </li><li> Enjuague la placa de Petri una vez con RPMI, a continuación, agregar el enjuague de los 70 filtros de micrón. </li><li> Con el extremo de caucho de un émbolo de una jeringa ml, moler las piezas restantes de médula ósea en la parte superior del filtro de 70 micras. </li><li> Lave el filtro una vez con RPMI. </li><li> Después de haberlo lavado, centrifugar la suspensión celular a 1200 rpm durante 5 minutos. </li><li> Lavar el precipitado celular mediante resuspensión en PBS. </li><li> Contar las células utilizando un hemocitómetro. Por lo general obtener ~ 90 millones de células de los huesos de un ratón (dos fémures más tibias dos). Después de contar las células, que centrifuga de nuevo a 1200 rpm durante 5 minutos. </li><li> Resuspender las células en una pequeña cantidad de RPMI y alícuota en los frascos de cultivo de tejido que contiene suficiente RPMI para que la concentración final es de 1 millón de células por ml. </li><li> Añadir citoquinas para promover el desarrollo de su tipo de célula de interés. En este caso, la citoquina interleuquina-3 (IL-3 = 10 ng / mL) para promover el desarrollo de los basófilos y mastocitos. Para adquirir los basófilos, incubar durante 10 días. De los mastocitos, se incuba durante 5 semanas. Cuando la generación de los mastocitos, los medios de comunicación e IL-3 se debe cambiar una vez por semana. </li><li> Aquí está una mirada a cómo las células del mástil debe aparecer justo después de la siembra. La imagen muestra la diferenciación de las células de la médula mástil en los basófilos y mastocitos 10 días después de la adición de la interleucina-3. </li><li> Una vez que el tipo celular deseado, proceda con el paso de la electroporación. </li></ol> Las células Electroporating <ol><li> Para comenzar la etapa de la electroporación, determinar el número de células en el frasco de cultivo. </li><li> Las células son típicamente electroporated a una densidad de 10 millones por ml, por lo que la transferencia del número de células necesarias para tubos cónicos y centrifugar los tubos a 1200 rpm durante 5 minutos. </li><li> Después de centrifugar, aspirar los medios de comunicación, se lavan las células con PBS 1X, y centrifugar nuevamente a 1200 rpm durante 5 minutos. </li><li> Aspirar el PBS y añadir Bio-Rad Gene tampón de electroporación generador de impulsos para hacer una suspensión celular de 10 millones de células por ml. </li><li> Después de la resuspensión de las células, agregar el ADN del plásmido deseado a una concentración final de 10 a 20 microgramos por mL. </li><li> Alícuota de 150 microlitros de la suspensión de células en los pozos de su elección en una placa de electroporación de 96 pozos. </li><li> Coloque la placa de electroporación en la cámara de placa MXcell y cierre la tapa. </li><li> Antes de la transfección de células cebadas, un protocolo de electroporación debe ser programado en el MXcell a menos que use un protocolo preestablecido o almacenados. A través de experimentos de optimización que hemos descubierto que la mayor eficiencia de la transfección de células cebadas se producen utilizando los protocolos de la onda de pulso cuadrado. Vamos a variar las condiciones de electroporación en la placa para entregar 300V/20ms, 350/15ms, 350V/20ms y 350V/10ms pulsos de onda cuadrada a 2000uF y 1000 ohmios. Una vez que el protocolo se ha establecido y se cargan en el dispositivo, pulse &quot;Pulse&quot; para electroporar las células. </li><li> Después de la electroporación es completa, la transferencia de las células en una placa de cultivo de tejidos. Por lo general la transferencia de cada muestra de 150 microlitros de electroporación de un pozo en una placa de tejido de 48 pocillos con 300 microlitros RPMI (complementado con un 10% de FCS, penicilina, estreptomicina, y beta-mercaptoetanol) a 10 nanogramos por mililitro de interleucina-3. Las células se incubaron durante la noche a 37 ° C, y luego analizaron 24 horas después de la expresión y la represión. </li></ol> TranResultados sfection La imagen de microscopía de fluorescencia de las células después de la electroporación con éxito utilizando los 20 microgramos por mL plásmido GFP se muestra en el video. Utilizando la electroporación MXcell eficiencia de transfección del sistema de alrededor del 30% se puede obtener. El sistema permite variar las condiciones para maximizar la eficiencia de transfección, mientras que el mantenimiento de la viabilidad celular.

A medida que más información sobre el genoma surge y nuevas herramientas como siRNA desarrollados, el uso de células fisiológicamente relevantes se ha convertido cada vez más importante para mejorar nuestra comprensión de los caminos de la enfermedad, las interacciones proteína-proteína, y la transducción de señales. Las células primarias se obtienen directamente de los tejidos o fluidos y cultivadas in vitro. Estas células se pueden manipular de muchas maneras, incluso mediante la introducción de material genético exógeno. Cada vez es más evidente que la forma más eficaz para introducir ADN plásmido o siRNA en las células primarias es la electroporación. Electroporación expone las células a pulsos eléctricos con el fin de aumentar de forma transitoria la permeabilidad de la membrana celular, permitiendo así que los ácidos nucleicos exógenos a entrar en la célula. Este método de transfección se puede optimizar para dar cabida a las diferencias entre los tipos de células diferentes / líneas y, por tanto, puede ser usado para transfectar las células cebadas, así como cualquier otro hueso derivadas de la médula de la célula. Además, a diferencia viral mediada por transfección, electroporación no impone límites en el tamaño de ADN transfectadas, ni requieren una preparación. A diferencia de los lípidos mediada por transfección, electroporación puede ser menos tóxico y no resulta en la captura endosomal del ácido nucleico transfectadas. Bio-Rad ha desarrollado el sistema de electroporación MXcell específicamente para estas células. la MXcell le permite variar las condiciones para maximizar su eficiencia de transfección y viabilidad de las células. Con el fin de optimizar la eficiencia de transfección y reducir al mínimo la muerte celular, una multitud de parámetros de electroporación, incluyendo voltaje, capacidad, resistencia y duración del impulso se puede ajustar y evaluar.

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the preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation

Cada vez es más evidente que la electroporación es la forma más eficaz para introducir ADN plásmido o siRNA en células primarias. El sistema de electroporación Gene Emisores MXcell y Gene tampón de electroporación Emisor se han desarrollado específicamente para transfectar ácidos nucleicos en células de mamíferos y difíciles de transfectar células, como el vídeo cells.This primaria y madre muestra cómo establecer cultivos primarios de células hematopoyéticas de la médula ósea murina , y luego a prepararse para la electroporación en el sistema de MXcell. Comenzamos por el aislamiento del fémur y la tibia. La médula ósea de ambos fémur y la tibia se recolectan y las culturas establecidas. Cultivos de células de médula ósea son entonces transfectadas y analizados.

Watch this Scientific Journal Video about The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation at JoVE.com

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

Este procedimiento se describe cómo establecer cultivos primarios de células hematopoyéticas de la médula ósea murina y es seguida por la transfección utilizando el sistema de electroporación Gene Emisores MXCell.

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 ...

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Research

JoVE Journal

Biology

25.6K vistas.  Bio-Rad Laboratories, Inc.  ChineseVersion 

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

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivación de células madre hematopoyéticas de células madre embrionarias murinas

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Investigación

Biología

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Cultivos neuronales primarios del cerebro de Drosophila pupas etapa tardía

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Electroporación de micobacterias

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

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 ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria disociada Mesencéfalo culturas dopamina celulares de roedores recién nacidos

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

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 ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primaria y electroporación plásmido del órgano de Corti murino.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Cultivos de células primarias de Drosophila Gástrula embriones

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Preparación de Primaria Myogenic precursor de célula / cultivos de mioblastos de basales Vertebrados Linajes

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

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 ...