JB è sostenuto da borse di studio dalle scienze naturali e ingegneria Research Council (NSERC). Il supporto per questo progetto è stato fornito dal Canadian Institutes of Health Research, Grant MOP-67066.

Questo protocollo è stato adattato da Lutz et al 1. Raccolta ed elaborazione di midollo osseo <ol><li> Isolamento della tibia e femore: eutanasia il mouse donatore e spruzzare le gambe con il 70% di etanolo. Afferrare la caviglia prima saldamente con una pinza smussato e cominciare a tagliare via la pelle e la muscolatura sottostante per esporre la tibia. Per evitare di danneggiare l&#39;osso, tagliato lentamente e in parallelo alla tibia, lasciando intatto il ginocchio. Per pulire il femore, immobilizzare l&#39;articolazione del ginocchio afferrando con una pinza contundente. Pulire via la muscolatura con un paio di forbici affilate e pinze curve. Continuare verso l&#39;alto fino a quando l&#39;anca è esposto e forbici può essere posizionato tra la testa del femore e dell&#39;anca. Togliere le ossa, tagliando tra comuni femore e dell&#39;anca e del luogo in tampone fosfato (PBS) su ghiaccio. </li><li> La rimozione del midollo osseo: Pulizia muscolatura residua dalle ossa con le forbici e pinze curve. Tagliare il epifisi di ogni osso e individuare la cavità centrale. Utilizzando una siringa da 10 ml caricato con PBS e un 25G0.5 &quot;ago, a livello del midollo osseo in un non-tessuto piatto rivestito di Petri. </li><li> L&#39;elaborazione di midollo osseo: Utilizzare l&#39;estremità in gomma dello stantuffo da una siringa da 1 ml per dissociare il midollo osseo in una cella singola sospensione (utilizzare un su e giù, non raschiando movimento). Raccogliere midollo osseo in un tubo conico falco, e risciacquare midollo osseo residuo con PBS. </li></ol> Coltura di cellule dendritiche <ol><li> Risospendere le cellule in DC media e precursori DC contare su un emocitometro (Figura 1). Vedrete le cellule di varie dimensioni, i precursori DC sono i più grandi e più brillanti. Differenziale contano solo queste cellule. Da due femori e due tibie di una wild-type C57BL / 6 del mouse (6-8 settimane), si dovrebbe aspettare tra 25-40 x 10 6 precursori DC totale. <img src="/files/ftp_upload/old/769_2008_6_30_3/article769_figure_1.jpg" alt="" width="218" height="217" /> Figura 1 </li><li> Celle di coltura ad una densità di 2 x 10 5 precursori DC / mL in DC dei media integrato con 40 ng / mL ricombinante murino GM-CSF. Cellule piastra in lastre di polistirene non tessuto rivestito di Petri. </li></ol> Aggiungi media (giorno 3) Per aggiornare i media, aggiungete la metà del volume totale di mezzi freschi integrata con 40 ng / mL GM-CSF. Sostituire un terzo della media (giorno 6) e la maturazione Per aggiornare i media, con attenzione rimuovere un terzo del volume totale dei media e sostituire questo volume con nuove mezzi di comunicazione integrata con DC 40 ng / mL GM-CSF al giorno 6 di cultura. Se lo si desidera, le cellule dendritiche possono essere stimolati per la maturazione con citochine o toll-like ligandi del recettore. Nel video, DC erano maturate dalla stimolazione durante la notte con 5 ng / ml CpG. Raccolta di cellule dendritiche Cultura DC è completa (Figura 2). Cellule saranno sia in sospensione e liberamente aderito alla piastra. Aderito cellule possono essere rimosse raschiando il piatto con un raschietto coltura di tessuti e risciacquo con PBS. Il numero totale di cellule aumenterà 5-8 volte durante la settimana di cultura e il periodo di differenziazione, quindi, si aspettano di raccogliere 1-1,6 x 10 6 cellule / ml. <img src="/files/ftp_upload/old/769_2008_6_30_5/article769_figure_2.jpg" alt="Figura 2" width="407" height="358" /> Figura 2 Reagenti <ol><li> DC dei media <ol><li> RPMI-1640 mezzi di comunicazione, integrato con: </li><li> 10% di siero fetale bovino </li><li> 100 UI / ml penicillina </li><li> 100 mg / ml streptomicina </li><li> 1% di L-glutammina </li><li> 0,1% 2-mercaptoetanolo </li><li> 1X aminoacidi non essenziali </li><li> 1X sodio piruvato </li></ol></li><li> Ricombinante murino GM-CSF <ol><li> rmGM-CSF (Peprotech inc, New Jersey, nessun catalogo. 315-03) </li></ol></li></ol>

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

DC sono utili per gli studi di interazioni immune innata e adattativa, e può essere impiegato come vettore di vaccino. In questo video, abbiamo dimostrato i passi per isolare midollo osseo e differenziare cellule dendritiche mieloidi in vitro. Dopo il periodo di coltura, queste cellule possono essere visualizzate sia le cellule al microscopio come aderenti, che spesso possiedono dendriti, e non aderente cellule rotonde. La DC può essere ulteriormente manipolata per la presentazione dell&#39;antigene da pulsante con l&#39;antigene o stimol...

<!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>recombinant murine GM-CSF</td>
<td>Reagent</td>
<td>Peptrotech</td>
<td>315-03</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

culture of myeloid dendritic cells from bone marrow precursors

Mieloide cellule dendritiche (DC) sono spesso usati per studiare le interazioni tra meccanismi immunitari innati e adattivi e la prima risposta alle infezioni. Perché questi sono l&#39;antigene più potenti cellule presentanti l&#39;, DC sono sempre più usato come vettore vaccino per studiare l&#39;induzione di risposta immunitaria antigene-specifica. In questo video, dimostriamo la procedura per la raccolta delle tibie e femori da un topo donatore, l&#39;elaborazione del midollo osseo e differenziare DC in vitro. Le proprietà del cambiamento DC a seguito di stimolazione: le cellule dendritiche immature sono fagociti potenti, mentre DC mature sono capaci di presentazione dell&#39;antigene e di interazione con CD4 + e CD8 + T. Questo cambiamento di attività funzionale corrisponde al upregulation dei marcatori di superficie delle cellule e la produzione di citochine. Molti agenti possono essere utilizzati per DC mature, tra cui citochine e Toll-like ligandi del recettore. In questo video, dimostriamo confronti citometria a flusso di espressione di due molecole co-stimolatorie, CD86 e CD40, e le citochine, IL-12, a seguito di stimolazione durante la notte con trattamento di CpG o modelli. Dopo la differenziazione, DC può essere ulteriormente manipolata per l&#39;utilizzo come vettore vaccino o per generare una risposta immunitaria antigene-specifica in vitro utilizzando impulsi peptidi o proteine, o trasdotte con vettori virali ricombinanti.

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

Culture of myeloid dendritic cells from bone marrow precursors

Questo video mostra la procedura per la differenziazione delle cellule dendritiche mieloidi dal midollo osseo dei topi. Isolamento di tibia e femore del mouse, e la lavorazione del midollo osseo sono dimostrati. Immagini che dimostrano morfologia cellulare prima e dopo la differenziazione, e le figure raffiguranti fenotipo delle cellule e IL-12 maturazione di produzione utilizzando i seguenti CpG sono mostrati.

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 ...

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

Research

JoVE Journal

Biology

22.4K Views.  McMaster University. Questo video mostra la procedura per la differenziazione delle cellule dendritiche mieloidi dal midollo osseo dei topi. Isolamento di tibia e femore del mouse, e la lavorazione del midollo osseo sono dimostrati. Immagini che dimostrano morfologia cellulare prima e dopo la differenziazione, e le figure raffiguranti fenotipo delle cellule e IL-12 maturazione di produzione utilizzando i seguenti CpG sono mostrati.

Video: Culture of myeloid dendritic cells from bone marrow precursors

Human ES cells: Starting Culture from Frozen Cells

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

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

Le cellule umane ES: Cultura partire da cellule congelate

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

Ricerca

Biologia

Culture of Mouse Neural Stem Cell Precursors

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

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

Cultura di cellule precursori staminali neurali del mouse

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

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

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

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

Generazione di cellule derivate dal midollo osseo dendritiche murini per l&#39;uso in 2-fotone Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La 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 ...

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 ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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

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

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

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

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

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 ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Espressione di proteine di fusione fluorescenti in macrofagi e cellule dendritiche derivate da midollo osseo Murine

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

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

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

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

Methods Article

Cultura di cellule dendritiche mieloidi dai precursori del midollo osseo