Harvesting Knochenmark aus Oberschenkel-und Schienbein <ol><li> Der erste Schritt des Verfahrens ist es, dem Knochenmark aus dem Oberschenkel-und Schienbein einer 6 Ernte - bis 12-Wochen alten Maus (wir sind mit BALB / c-Mäusen, aber Sie können diesen Vorgang mit jeder Maus-Stamm zu tun). Um zu beginnen, euthanize der Maus durch CO 2-Inhalation (wird nicht angezeigt). Ernte Oberschenkelknochen und Schienbein von den Hinterbeinen eines jeden. Dann, mit einer Rasierklinge geschnitten jedem Ende der Oberschenkel auf die Hüft-und Kniegelenke zu entfernen und bis auf die Knochen freizulegen. </li><li> In ähnlicher Weise geschnitten jedem Ende der Tibia, die Knie-und Sprunggelenk angrenzenden Regionen zu entfernen und bis auf die Knochen freizulegen. </li><li> Nehmen Sie ein 3-ml-Spritze mit einer 26-Gauge-Nadel und füllen es mit RPMI mit 10% FBS, Penicillin, Streptomycin und beta-Mercaptoethanol (BME = 100 uM) ergänzt. Mit Pinzette, halten Sie die Knochen über eine Petrischale mit RPMI-Medien. Legen Sie die Nadel der Spritze in das eine Ende des Knochens und drücken Sie den Kolben zu spülen, das Knochenmark. Die Kanüle kann auf und ab bewegt im Inneren der Knochen zu spülen verbleibende Knochenmark werden. </li><li> Wiederholen Sie den Vorgang für alle die Knochen, und fahren Sie dann mit Gründung der Kulturen. </li></ol> Gründung Kulturen <ol><li> Zum Aufbau der Knochenmark-Kulturen, die Pipette gespült Knochenmark nach oben und unten mehrmals zum Aufbrechen der Gewebe in einer Zellsuspension. </li><li> Legen Sie einen 70-Mikron-Filter auf einem 50 mL konischen Rohr, und fügen Sie die Zellsuspension auf den Filter. </li><li> Spülen Sie die Petrischale einmal mit RPMI, fügen Sie dann die zu den 70-Mikron-Filter zu spülen. </li><li> Mit dem Gummi Ende einer 1 mL Spritzenkolben, schleifen das Knochenmark Stücke übrigen auf der 70-Mikron-Filter. </li><li> Waschen Sie den Filter einmal mit RPMI. </li><li> Nach dem Waschen der Filter, Zentrifugen der Zellsuspension bei 1200 rpm für 5 Minuten. </li><li> Waschen Sie die Zellpellet durch Resuspendieren in PBS. </li><li> Zählen Sie die Zellen mit Hilfe einer Zählkammer. Wir in der Regel erhalten ~ 90 Millionen Zellen von einer Maus die Knochen (zwei Oberschenkelknochen sowie zwei tibiae). Nach Auszählung der Zellen, Zentrifuge sie wieder bei 1200 rpm für 5 Minuten. </li><li> Resuspendieren der Zellen in eine kleine Menge RPMI und aliquoten sie in Zellkulturflaschen mit RPMI genug, so dass die Endkonzentration 1 Million Zellen pro ml ist. </li><li> Add Zytokine auf die Entwicklung Ihres Zelltyp von Interesse zu fördern. In diesem Fall ist das Zytokin Interleukin-3 (IL-3 = 10 ng / mL) zugegeben, um die Entwicklung von Basophilen und Mastzellen zu fördern. Zum Erwerb Basophile, für 10 Tage bebrüten. Für Mastzellen, für 5 Wochen bebrüten. Bei der Generierung von Mastzellen, die Medien und IL-3 sollte einmal pro Woche gewechselt werden. </li><li> Hier ist ein Blick darauf, wie Mastzellen sollten unmittelbar nach der Beschichtung auftreten. Dieses Bild zeigt die Differenzierung der Mast Knochenmarkzellen in Basophilen und Mastzellen 10 Tage nach der Zugabe von Interleukin-3. </li><li> Sobald die gewünschten Zelltyp erhalten, mit der Elektroporation Schritt fort. </li></ol> Elektroporation Cells <ol><li> Zu Beginn der Elektroporation Schritt bestimmen die Anzahl der Zellen in der Kulturflasche. </li><li> Cells sind typischerweise bei einer Dichte von 10 Millionen pro ml elektroporiert, so übertragen die erforderliche Zellzahl auf konische Röhrchen und zentrifugieren bei 1200 rpm für 5 Minuten. </li><li> Nach dem Zentrifugieren absaugen Medien, waschen Sie die Zellen mit 1X PBS, und Zentrifuge wieder bei 1200 rpm für 5 Minuten. </li><li> Saugen Sie das PBS und fügen Bio-Rad Gene Pulser Elektroporation Puffer, um eine Zellsuspension von 10 Millionen Zellen pro ml zu machen. </li><li> Nach dem Resuspendieren der Zellen, fügen Sie die gewünschten Plasmid-DNA in einer Endkonzentration von 10 bis 20 Mikrogramm pro Milliliter. </li><li> Aliquot 150 Mikroliter der Zellsuspension in die Vertiefungen Ihrer Wahl auf eine 96-Well Platte Elektroporation. </li><li> Legen Sie die Elektroporation Platte in der MXcell Platte Kammer und den Deckel schließen. </li><li> Vor der Transfektion von Mastzellen, muss eine Elektroporation Protokoll in die MXcell es sei denn, mit einem vorgegebenen oder gespeicherte Protokoll programmiert werden. Durch Optimierung Experimenten haben wir herausgefunden, dass die höchste Transfektionseffizienz von Mastzellen kommen mit Rechteckimpuls-Protokolle. Wir werden die Elektroporation Bedingungen auf der Platte zu variieren, um 300V/20ms, 350/15ms, 350V/20ms und 350V/10ms Rechteckimpulse bei 2000uF und 1000 Ohm zu liefern. Sobald das Protokoll festgelegt wurde und geladen auf das Gerät, drücken Sie &quot;Pulse&quot;, um die Zellen elektroporieren. </li><li> Nach der Elektroporation abgeschlossen ist, übertragen Sie die Zellen einer Gewebekultur Platte. Wir typischerweise Transfer je 150 Mikroliter Elektroporation Probe auf ein gut in einer 48-well Zellkultur-Platte mit 300 Mikroliter RPMI (ergänzt mit 10% FCS, Penicillin, Streptomycin und beta-Mercaptoethanol) mit 10 Nanogramm pro ml Interleukin-3. Die Zellen werden über Nacht bei 37 ° C, dann analysiert 24 Stunden später für die Expression und Unterdrückung. </li></ol> Transfection Ergebnisse Die Fluoreszenz-Mikroskopie Bild der Zellen nach erfolgreicher Elektroporation mit 20 Mikrogramm pro ml GFP-Plasmid ist in dem Video zu sehen. Mit dem MXcell Elektroporationssystem Transfektionseffizienz von ca. 30% erzielt werden kann. Das System ermöglicht Ihnen, die Bedingungen zu Ihrem Transfektionseffizienz zu maximieren variieren, dabei jedoch die Lebensfähigkeit der Zellen.

Da immer mehr genomische Information entsteht und neue Werkzeuge wie siRNA entwickelt werden, hat die Verwendung von physiologisch relevanten Zellen immer mehr an Bedeutung für unser Verständnis der Krankheit Wege, Protein-Protein-Interaktionen und Signaltransduktion weiter. Primäre Zellen werden direkt von Geweben oder Flüssigkeiten gewonnen und in vitro kultiviert. Diese Zellen können in eine Reihe von Möglichkeiten, unter anderem durch die Einführung von exogenen genetischen Materials verändert werden. Es wird immer offensichtlicher, dass der effektivste Weg, um Plasmid-DNA oder siRNA in primäre Zellen einzuschleusen Elektroporation ist. Elektroporation macht Zellen in elektrische Impulse um vorübergehend erhöhen die Durchlässigkeit der Zellmembran, wodurch exogene Nukleinsäuren in die Zelle eindringen. Diese Methode der Transfektion optimiert, um die Unterschiede zwischen verschiedenen Zelltypen / Leitungen und somit können an Mastzellen transfizieren werden sowie alle anderen Knochenmark stammende Zellen unterzubringen. Außerdem, im Gegensatz viral-vermittelte Transfektion, Elektroporation ist nicht Grenzen für die Größe der transfizierten DNA, noch erfordert umfangreiche Vorbereitungen. Im Gegensatz zu Lipid-vermittelte Transfektion, Elektroporation kann weniger toxisch sein und nicht in endosomalen Abfangen der transfizierten Nukleinsäure führen. Bio-Rad hat die MXcell Elektroporation, das speziell für diese Zellen entwickelt. die MXcell können Sie variieren, um Ihre Transfektionseffizienz und die Lebensfähigkeit der Zellen zu maximieren. Um die Transfektionseffizienz zu optimieren und Zelltod, eine Vielzahl von Elektroporation Parameter einschließlich Spannung, können Kapazität, Widerstand, und Pulslänge eingestellt und bewertet werden.

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

Es wird immer deutlicher, dass die Elektroporation der effektivste Weg, um Plasmid-DNA oder siRNA in primären Zellen einzuführen. Die Gene Pulser MXcell Elektroporation und Gene Pulser Elektroporation Puffer wurden speziell entwickelt, um Nukleinsäuren in Säugerzellen und schwer zu transfizieren Zellen, wie Primär-und Stammzellen cells.This Video transfizieren demonstriert, wie primäre hämatopoetischen Zellkulturen aus Knochenmark der Maus zu etablieren , und dann bereiten sie für die Elektroporation in den MXcell System. Wir beginnen mit der Isolierung von Ober-und Unterschenkels. Knochenmark aus beiden Ober-und Unterschenkels werden dann geerntet und Kulturen etabliert. Cultured Knochenmarkzellen werden dann transfiziert und analysiert.

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

Dieses Verfahren beschreibt, wie primäre hämatopoetischen Zellkulturen aus Knochenmark der Maus zu etablieren und wird durch Transfektion mit dem Gene Pulser MXCell Elektroporation Systems.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

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 Aufrufe.  Bio-Rad Laboratories, Inc.  ChineseVersion 

Serie: 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.

Ableitung von hämatopoetischen Stammzellen aus embryonalen Stammzellen der Maus

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 ...

Forschung

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.

Primäre neuronale Kulturen aus den Gehirnen von Late Stage Drosophila Puppen

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.

Elektroporation von Mykobakterien

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.

Kultur myeloide dendritische Zellen aus dem Knochenmark Vorstufen

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.

Primäre Dissoziierte Mittelhirn Dopamin Zellkulturen aus Nager Neugeborene

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 von hämatopoetischen Zellen im Knochenmark

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.

Primäre Kultur und Plasmid Elektroporation der Murine Corti-Organ.

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.

Primären Zellkulturen aus Drosophila Gastrula Embryonen

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.

Herstellung von primären Myogenic Vorläuferzelle / Myoblastenkulturen von Basal Wirbelstammbaum

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.

Osteoklasten Ableitung aus Knochenmark der Maus

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