La récolte de moelle osseuse provenant fémurs et tibias <ol><li> La première étape de la procédure est de récolter la moelle osseuse dans le fémur et le tibia d&#39;un 6 - à 12 semaines vieille souris (nous utilisons souris Balb / c, mais vous pouvez faire cette procédure avec toute souche de souris). Pour commencer, euthanasier la souris par inhalation de CO 2 (ne sera pas montré). Fémur et tibias Récolte de la pattes postérieures de chacun. Puis, avec une lame de rasoir, couper chaque extrémité des fémurs d&#39;enlever la hanche et du genou et d&#39;exposer la moelle. </li><li> De la même manière, couper chaque extrémité du tibia pour enlever le genou et la cheville régions adjacentes et d&#39;exposer la moelle. </li><li> Prendre une seringue de 3 ml avec une aiguille de calibre 26 et de le remplir avec RPMI supplémenté avec 10% de FBS, la pénicilline, la streptomycine et le bêta-mercaptoéthanol (BME = 100 UM). En utilisant une pince à épiler, tenez l&#39;os au cours d&#39;une boîte de Petri contenant un milieu RPMI. Insérez l&#39;aiguille de la seringue dans une extrémité de l&#39;os et appuyer sur le piston pour chasser la moelle osseuse. L&#39;aiguille de la seringue peut être déplacé de haut en bas dans l&#39;os pour débusquer la moelle résiduelle. </li><li> Répétez la procédure pour tous les os, puis procéder à l&#39;établissement des cultures. </li></ol> Établir des cultures <ol><li> Pour établir les cultures de moelle osseuse, une pipette de la moelle osseuse rincée de haut en bas plusieurs fois pour briser le tissu dans une suspension cellulaire. </li><li> Placer un filtre de 70 microns au-dessus d&#39;un tube conique de 50 ml, et ajouter la suspension de cellules sur le filtre. </li><li> Rincez les boîtes de Pétri une fois avec du RPMI, puis ajouter l&#39;eau de rinçage à l&#39;filtre 70 microns. </li><li> Utilisation de l&#39;extrémité en caoutchouc d&#39;un plongeur 1 ml seringue, broyer les morceaux de moelle osseuse restent sur le dessus du filtre de 70 microns. </li><li> Lavez le filtre une fois avec du RPMI. </li><li> Après le lavage du filtre, centrifuger les cellules en suspension à 1200 rpm pendant 5 minutes. </li><li> Laver le culot cellulaire par remise en suspension dans du PBS. </li><li> Compter les cellules en utilisant un hématimètre. Nous obtiennent généralement ~ 90 millions de cellules à partir des os d&#39;une souris (deux fémurs et deux tibias). Après avoir compté les cellules, les centrifuger à 1200 rpm pendant 5 minutes. </li><li> Resuspendre les cellules dans une petite quantité de RPMI et aliquot eux dans des flacons de culture de tissus contenant suffisamment de RPMI sorte que la concentration finale est de 1 million de cellules par mL. </li><li> Ajouter des cytokines de promouvoir le développement de votre type de cellule d&#39;intérêt. Dans ce cas, la cytokine interleukine-3 (IL-3 = 10 ng / ml) est ajoutée à promouvoir le développement des basophiles et les mastocytes. Pour acquérir les basophiles, incuber pendant 10 jours. Pour les mastocytes, les incuber pendant 5 semaines. Lors de la génération des mastocytes, les médias et l&#39;IL-3 devrait être changé une fois par semaine. </li><li> Voici un aperçu de la façon dont les mastocytes devrait apparaître juste après placage. Cette image montre la différenciation des cellules de la moelle mât dans les basophiles et les mastocytes 10 jours après l&#39;ajout d&#39;interleukine-3. </li><li> Une fois le type de cellule désiré est obtenu, passez à l&#39;étape électroporation. </li></ol> Cellules électroporation <ol><li> Pour commencer l&#39;étape d&#39;électroporation, déterminer le nombre de cellules dans le flacon de culture. </li><li> Les cellules sont généralement électroporation à une densité de 10 millions par ml, alors le transfert le nombre de cellules nécessaires pour tubes coniques et centrifuger les tubes à 1200 tpm pendant 5 minutes. </li><li> Après centrifugation, aspirer les médias, se laver les cellules avec du PBS 1X et centrifuger à nouveau à 1200 rpm pendant 5 minutes. </li><li> Aspirer le PBS et ajouter de Bio-Rad tampon Gene Pulser électroporation de faire une suspension cellulaire de 10 millions de cellules par mL. </li><li> Après remise en suspension des cellules, ajouter de l&#39;ADN plasmidique désiré à une concentration finale de 10 à 20 microgrammes par ml. </li><li> Aliquoter 150 microlitres de la suspension cellulaire dans des puits de votre choix sur une plaque de 96 puits électroporation. </li><li> Mettez la plaque électroporation dans la chambre de la plaque MXcell et fermer le couvercle. </li><li> Avant de transfection de cellules de mât, un protocole d&#39;électroporation doit être programmé dans le MXcell à moins d&#39;utiliser un protocole prédéfini ou stockées. Grâce à des expériences d&#39;optimisation que nous avons découvert que l&#39;efficacité de la transfection de cellules haute du mât se produisent en utilisant des protocoles carrés onde de pouls. Nous allons modifier les conditions d&#39;électroporation sur la plaque de livrer 300V/20ms, 350/15ms, 350V/20ms, et des impulsions d&#39;ondes carrées 350V/10ms au condensateur 2000uF et 1000 ohms. Une fois le protocole a été établi et chargé sur le périphérique, appuyez sur «Pulse» pour électroporer les cellules. </li><li> Après électroporation est terminée, le transfert des cellules sur une plaque de culture de tissus. Nous généralement transférer chaque échantillon de 150 microlitre électroporation à un puits dans une plaque de 48 puits de culture de tissu contenant 300 microlitres RPMI (complété avec 10% de FCS, la pénicilline, la streptomycine et le bêta-mercaptoéthanol) avec 10 nanogrammes par ml d&#39;interleukine-3. Les cellules sont incubées une nuit à 37 ° C, puis dosée 24 heures plus tard, d&#39;expression et de répression. </li></ol> TranRésultats sfection L&#39;image de microscopie à fluorescence des cellules après l&#39;électroporation réussie en utilisant 20 microgrammes par ml GFP plasmide est montré dans la vidéo. Utilisation de l&#39;efficacité MXcell électroporation système de transfection de l&#39;ordre de 30% peut être obtenue. Le système vous permet de varier les conditions afin de maximiser votre efficacité de la transfection, tout en maintenant la viabilité cellulaire.

Comme plus d&#39;informations génomiques émerge et de nouveaux outils tels que siARN sont développés, l&#39;utilisation de cellules physiologiquement pertinente est devenue de plus en plus important d&#39;approfondir notre compréhension des voies de la maladie, interactions protéine-protéine, et la transduction du signal. Les cellules primaires sont obtenues directement à partir de tissus ou les liquides et cultivées in vitro. Ces cellules peuvent être manipulés dans un certain nombre de façons, notamment par l&#39;introduction de matériel génétique exogène. Il est de plus en plus évident que la façon la plus efficace pour introduire de l&#39;ADN plasmidique ou siRNA dans des cellules primaires est l&#39;électroporation. L&#39;électroporation expose les cellules à des impulsions électriques de manière à transitoirement augmenter la perméabilité de la membrane cellulaire, permettant ainsi aux acides nucléiques exogènes pour entrer dans la cellule. Cette méthode de transfection peut être optimisée pour accueillir les différences entre les différents types de cellules / lignes et, par conséquent, peuvent être utilisés pour transfecter des cellules de mât ainsi que tout autre os de cellules dérivées de la moelle. Par ailleurs, contrairement virale médiée par transfection, l&#39;électroporation n&#39;impose pas de limites sur la taille de l&#39;ADN transfecté, ni exiger une préparation importante. Contrairement aux lipides transfection médiée, l&#39;électroporation peut être moins toxique et n&#39;entraîne pas de piégeage de l&#39;acide endosomal transfectées nucléiques. Bio-Rad a développé le système d&#39;électroporation MXcell spécifiquement pour ces cellules. l&#39;MXcell vous permet de varier les conditions afin de maximiser votre efficacité de la transfection et la viabilité cellulaire. Afin d&#39;optimiser l&#39;efficacité de transfection et de minimiser la mort cellulaire, une multitude de paramètres, y compris l&#39;électroporation de tension, capacité, résistance et durée d&#39;impulsion peut être réglée et évalués.

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

Il est de plus en plus évident que l&#39;électroporation est le moyen le plus efficace pour introduire de l&#39;ADN plasmidique ou siRNA dans des cellules primaires. Le système d&#39;électroporation Gene Pulser MXcell et tampon d&#39;électroporation Gene Pulser ont été spécifiquement développés pour transfecter des acides nucléiques dans les cellules de mammifères et difficiles à transfecter des cellules, telles que la vidéo cells.This primaires et la tige montre comment mettre en place des cultures primaires de cellules hématopoïétiques de la moelle osseuse de souris , puis les préparer pour l&#39;électroporation dans le système MXcell. Nous commençons par isoler le fémur et le tibia. La moelle osseuse des deux fémur et le tibia sont ensuite récoltées et les cultures sont établies. Des cultures de cellules de moelle osseuse sont ensuite transfectées et analysées.

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

Cette procédure décrit comment établir des cultures primaires de cellules hématopoïétiques de la moelle osseuse de souris et est suivie par transfection en utilisant le système d&#39;électroporation Gene Pulser MXCell.

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

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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Biology

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

Vidéo: 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.

Dérivation de cellules souches hématopoïétiques de cellules souches embryonnaires murines

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 ...

Recherche

Biologie

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.

Cultures neuronales primaires du cerveau des drosophiles stade tardif pupes

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.

Electroporation de mycobactéries

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.

Culture de cellules dendritiques myéloïdes à partir de précurseurs de moelle osseuse

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.

Primaire Dissocié cultures Mésencéphale dopamine cellulaire du nouveau-né contre les rongeurs

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.

Homing des cellules hématopoïétiques de la moelle osseuse

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.

Culture primaire et l&#39;électroporation plasmidique de l&#39;Organe de Corti murin.

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.

Cultures de cellules primaires à partir Drosophile Embryons Gastrula

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.

Préparation du primaire Myogenic précurseurs cellulaires / cultures de myoblastes de basales vertébrés lignées

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.

Ostéoclastes Dérivation de moelle osseuse de souris

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