La raccolta di midollo osseo da femori e tibie <ol><li> Il primo passo della procedura è quello di raccogliere midollo osseo dal femore e la tibia di un 6 - a 12 settimane vecchio mouse (stiamo usando topi BALB / c, ma si può fare questa procedura con qualsiasi ceppo di topi). Per iniziare, eutanasia il mouse da CO 2 inalazioni (non sarà mostrato). Vendemmia femore e tibia dalle zampe posteriori di ciascuno. Poi, con una lametta da barba, tagliare ogni estremità del femori per rimuovere l&#39;anca e le articolazioni del ginocchio e per esporre il midollo. </li><li> In modo simile, tagliare ogni estremità della tibia per rimuovere il ginocchio e della caviglia regioni limitrofe e per esporre il midollo. </li><li> Fate una siringa da 3 ml con una 26-gauge e riempirlo con RPMI supplementato con 10% FBS, penicillina, streptomicina, e beta-mercaptoetanolo (BME = 100 uM). Utilizzando una pinzetta, tenere l&#39;osso su una piastra di Petri contenente RPMI media. Inserire l&#39;ago della siringa in una delle estremità delle ossa e premere lo stantuffo per scovare il midollo osseo. L&#39;ago della siringa può essere spostato su e giù dentro l&#39;osso per scovare midollo residuo. </li><li> Ripetere la procedura per tutte le ossa, e quindi procedere con istituisce la culture. </li></ol> Stabilire Culture <ol><li> Per stabilire le culture del midollo osseo, pipetta il midollo osseo lavata su e giù parecchie volte per rompere il tessuto in una sospensione cellulare. </li><li> Collocare un filtro di 70 micron in cima ad un tubo 50 ml conica, e aggiungere la sospensione cellulare al filtro. </li><li> Sciacquare la piastra Petri una volta con RPMI, quindi aggiungere il risciacquo del filtro per i 70 micron. </li><li> Utilizzando la fine di gomma di una siringa da 1 ml stantuffo, macinare i pezzi rimanenti del midollo osseo nella parte superiore del filtro di 70 micron. </li><li> Lavare il filtro una volta con RPMI. </li><li> Dopo aver lavato il filtro, centrifuga la sospensione cellulare a 1200 rpm per 5 minuti. </li><li> Lavare il pellet da risospendere in PBS. </li><li> Contare le cellule utilizzando un emocitometro. Noi di solito ottenere ~ 90 milioni di cellule di ossa di un mouse (due femori e due tibie). Dopo il conteggio delle cellule, li centrifuga di nuovo a 1200 rpm per 5 minuti. </li><li> Risospendere le celle di una piccola quantità di RPMI e le aliquote in fiasche colture di tessuti contenenti abbastanza RPMI in modo che l&#39;concentrazione finale è di 1 milione di cellule per mL. </li><li> Aggiungi citochine a promuovere lo sviluppo del vostro tipo di cellula di interesse. In questo caso, la citochina interleuchina-3 (IL-3 = 10 ng / mL) viene aggiunto per promuovere lo sviluppo dei basofili e mastociti. Per acquisire basofili, incubare per 10 giorni. Per i mastociti, incubare per 5 settimane. Quando si genera mastociti, i media e IL-3 dovrebbe essere cambiata una volta alla settimana. </li><li> Ecco uno sguardo a come mastociti dovrebbe apparire subito dopo placcatura. Questa immagine mostra la differenziazione delle cellule del midollo dell&#39;albero in basofili e mastociti 10 giorni dopo l&#39;aggiunta di interleuchina-3. </li><li> Una volta che il tipo cellulare desiderato si ottiene, procedere con il passaggio elettroporazione. </li></ol> Cellule Electroporating <ol><li> Per iniziare il passo elettroporazione, determinare il numero di cellule nel pallone cultura. </li><li> Le cellule sono in genere elettroporate ad una densità di 10 milioni per ml, in modo da trasferire il numero di cellule per tubi conici e centrifugare le provette a 1200 rpm per 5 minuti. </li><li> Dopo la centrifugazione, aspirare i media, lavare le cellule con PBS 1X, e centrifugare di nuovo a 1200 rpm per 5 minuti. </li><li> Aspirare il PBS e aggiungere Bio-Rad Gene buffer di elettroporazione Pulser per fare una sospensione di cellule di 10 milioni di cellule per ml. </li><li> Dopo risospendere le cellule, aggiungere il DNA desiderato plasmide ad una concentrazione finale di 10 a 20 microgrammi per ml. </li><li> Aliquota 150 microlitri di sospensione cellulare in pozzi di vostra scelta su un piatto elettroporazione 96-bene. </li><li> Mettete la placca nella camera di elettroporazione MXcell piastra e chiudere il coperchio. </li><li> Prima di trasfezione dei mastociti, un protocollo di elettroporazione deve essere programmato in MXcell a meno di utilizzare un protocollo predefinito o memorizzati. Attraverso esperimenti di ottimizzazione che hanno scoperto che la più alta efficienza di trasfezione dei mastociti avvenire utilizzando protocolli di piazza dell&#39;onda di polso. Noi variare le condizioni elettroporazione sul piatto per fornire 300V/20ms, 350/15ms, 350V/20ms e 350V/10ms impulsi a onda quadra 2000uF e 1000 ohm. Una volta che il protocollo è stato impostato e caricata sul dispositivo, premere il tasto &quot;Pulse&quot; per electroporate le cellule. </li><li> Dopo l&#39;elettroporazione è completa, trasferire le cellule a una piastra di coltura dei tessuti. Noi di solito trasferire ogni campione elettroporazione 150 microlitri di un pozzo in un 48-pozzetti di coltura contenente 300 microlitri RPMI (supplementato con 10% FCS, penicillina, streptomicina, e beta-mercaptoetanolo) con 10 nanogrammi per ml interleuchina-3. Le cellule vengono incubate overnight a 37 ° C, poi analizzati 24 ore più tardi per l&#39;espressione e la soppressione. </li></ol> Transfection Risultati L&#39;immagine di microscopia a fluorescenza delle cellule dopo l&#39;elettroporazione di successo con 20 microgrammi per ml GFP plasmide viene mostrato nel video. Utilizzando il sistema di elettroporazione MXcell efficienze di trasfezione di circa il 30% può essere ottenuto. Il sistema consente di variare le condizioni per massimizzare l&#39;efficienza di trasfezione, pur mantenendo la vitalità cellulare.

Come più informazioni genomiche emerge e nuovi strumenti come siRNA sono sviluppati, l&#39;uso di cellule fisiologicamente rilevanti è diventato sempre più importante per migliorare la nostra comprensione delle vie di malattia, interazioni proteina-proteina, e la trasduzione del segnale. Pile sono ottenute direttamente da tessuti o fluidi e coltivate in vitro. Queste cellule possono essere manipolati in vari modi, anche attraverso l&#39;introduzione di materiale genetico esogeno. E &#39;sempre più evidente che il modo più efficace per introdurre DNA plasmidico o siRNA in cellule primarie è elettroporazione. Elettroporazione espone le cellule a impulsi elettrici al fine di aumentare temporaneamente la permeabilità della membrana cellulare, consentendo in tal modo esogeno acidi nucleici per entrare nella cellula. Questo metodo di trasfezione può essere ottimizzato per accogliere le differenze tra diversi tipi di cellule / linee e, quindi, può essere utilizzato per trasfettare mastociti così come qualsiasi altro osso cellule derivate dal midollo. Inoltre, a differenza virale mediata trasfezione, elettroporazione non impone limiti alle dimensioni del DNA transfettate, né richiedono una preparazione. A differenza dei lipidi mediata trasfezione, elettroporazione può essere meno tossico e non dia luogo a cattura endosomiali degli acidi nucleici transfettate. Bio-Rad ha sviluppato il sistema di elettroporazione MXcell appositamente per queste cellule. il MXcell consente di variare le condizioni per massimizzare l&#39;efficienza di trasfezione e la vitalità cellulare. Al fine di ottimizzare l&#39;efficienza di trasfezione e ridurre al minimo la morte delle cellule, una moltitudine di parametri tra cui elettroporazione tensione, capacità, resistenza e durata dell&#39;impulso può essere regolata e valutati.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gene Pulser MXcell Electroporation System</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2670</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser Electroporation Buffer</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2677</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

E &#39;sempre più evidente che elettroporazione è il modo più efficace per introdurre DNA plasmidico o siRNA in cellule primarie. Il gene Pulser sistema di elettroporazione MXcell e Gene buffer di elettroporazione Pulser sono stati specificamente sviluppati per trasfettare acidi nucleici nelle cellule dei mammiferi e di difficile trasfezione delle cellule, come i video cells.This primarie e staminali dimostra come stabilire colture primarie di cellule emopoietiche dal midollo osseo murino , e poi li preparano per l&#39;elettroporazione nel sistema MXcell. Si comincia isolando femore e della tibia. Midollo osseo da femore e della tibia vengono poi raccolte e le culture sono stabiliti. Colture di cellule del midollo osseo sono poi transfettate e analizzati.

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

Questa procedura descrive come stabilire colture primarie di cellule emopoietiche dal midollo osseo murino ed è seguita dalla trasfezione con il gene Pulser sistema di elettroporazione MXCell.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

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

<a class="grey_button" href="/video/1047">
ChineseVersion
</a>

the-preparation-primary-hematopoietic-cell-cultures-from-murine-bone

Research

JoVE Journal

Biology

25.6K Views.  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.

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

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 ...

Ricerca

Biologia

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.

Culture primarie neuronale dal cervello di tardo fase 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 ...

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.

Elettroporazione di micobatteri

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 di cellule dendritiche mieloidi dai precursori del midollo osseo

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 dissociato mesencefalo colture cellulari dopamina da roditori neonati

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 delle cellule ematopoietiche del midollo osseo

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 e elettroporazione plasmidi dell&#39;Organo murini di 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 ...

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.

Colture cellulari primarie da Drosophila Gastrula embrioni

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.

Preparazione di primaria Miogenica precursori cellulari / Culture mioblasti dal basale vertebrati Lignaggi

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.

Osteoclasti derivazione dal midollo osseo di topo

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