Préparation des neurones de rat fœtal E18 corticales pour compartimentation Avant de commencer, il est important de réchauffer tous les médias et les réactifs nécessaires à 37 ° C. Il est également important de stériliser tout ce qui est utilisé pour la préparation des cellules (par exemple, les ampoules de caoutchouc, supports pour tubes, des bouteilles médias, etc), et celui qui est placé dans la hotte, en essuyant avec de l&#39;éthanol à 70%. <ol><li> Placez deux morceaux de cortex de rat fœtal E18 (un cerveau, précédemment disséqué) dans un tube de 15 ml contenant 1 ml glacée sans calcium magnésium tampon sans dissection. </li><li> Ajouter 1 ml de 0,25% de trypsine-EDTA au cortex dans le tampon de la dissection, portant le volume final de 2 ml et la concentration de trypsine finale à 0,125%. </li><li> Placez le tube de 15 ml dans un bain d&#39;eau à 37 ° C pendant 8 minutes. </li><li> Pendant ce temps, le feu polonaise de verre 3 pipettes Pasteur, formant des ouvertures plus petites successivement, dans une enceinte de biosécurité pour aider à maintenir la stérilité. </li><li> Après l&#39;incubation 8 minutes du cortex, ajouter 10 ml de DMEM contenant 10% de FBS au cortex pour aider à arrêter la réaction trypsine. </li><li> Centrifuger le tube de 15 ml contenant du cortex et DMEM/10% de FBS à 2500 rpm pendant 2 minutes. </li><li> Dans le cabinet de biosécurité, éliminer le surnageant du cortex en utilisant une pipette Pasteur en verre avec des ventouses ci-joint. Soyez prudent de ne pas déranger ou déloger le culot. </li><li> Ajouter 1 ml de NMB à la pastille cortex et délicatement la pipette de haut en bas. Il est très important pour éviter de créer des bulles d&#39;air tout en haut et en bas de pipetage, comme les bulles d&#39;air peuvent endommager les cellules par oxydation. </li><li> Utilisez le feu poli pipette Pasteur avec la plus large ouverture de triturer le cortex en attachant une poire en caoutchouc stériles jusqu&#39;à la fin et pipetage haut et en bas 5 fois, encore une fois en faisant attention à ne pas introduire de bulles d&#39;air. Continuez ce processus avec les 2 autres pipettes en verre, chacun avec une taille d&#39;ouverture diminué. </li><li> Après trituration, centrifuger le bas des cellules de nouveau à 2500 rpm pendant 2 minutes. </li><li> Après centrifugation, une fois de retirer le surnageant et remettre le culot cellulaire dans 2 ml de MNB. </li><li> Filtrer la solution de cellules en suspension à travers un tamis 45 cellules um. </li><li> Colorer les cellules au bleu trypan, compté, et chargé dans les appareils. En général, nous charge de 20 ul de cellules par l&#39;appareil. </li></ol> Remarque: la concentration finale de cellules est typiquement comprise entre 2,5 millions de cellules / ml et 8 millions de cellules / ml Chargement des cellules Après que les cellules ont été comptées, il est temps de charger les cellules de l&#39;appareil: <ol><li> Apportez les dispositifs précédemment préparés contenant MNB dans l&#39;armoire de biosécurité afin de maintenir la stérilité. </li><li> Retirer l&#39;excès des médias provenant des puits par aspiration sous vide. Cependant, soyez prudent pour éviter d&#39;enlever tous les médias - il doit y avoir certains médias dans le chenal principal. </li><li> Appliquer 20 ul de cellules dans le réservoir en haut à gauche de l&#39;appareil (voir schéma si nécessaire). Le flux de cellules dans l&#39;appareil et fixer à la surface traitée PLL. </li><li> Après le chargement, placer les appareils contenant des cellules de retour dans un incubateur pendant 10 minutes pour permettre aux cellules d&#39;attacher. </li><li> Après 10 minutes, remplir les réservoirs avec les médias et les dispositifs replacer dans l&#39;incubateur. </li></ol>

<ol>
	<li>Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nat Protoc. 1, 2128-2136 (2006).</li><li>Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nat Methods. 2, 599-605 (2005).</li></ol>

Densités cellulaires peuvent être variés en fonction de la demande. Toutefois, l&#39;ensemencement des neurones primaires à trop basse d&#39;une densité entraîne généralement la mort cellulaire. Selon l&#39;incubateur et le niveau d&#39;humidité, les médias peuvent avoir besoin d&#39;être changé dans les appareils tous les 2 à 3 jours. Lors du changement de support, il est important après la suppression des médias à partir des réservoirs, de ne jamais retirer le support des canaux principaux. En outre, il est conseillé de placer des milieux frais dans le puits supérieur et permettre à certains milieux frais de circuler à travers l&#39;appareil et dans les principaux canaux avant de remplir tous les réservoirs.

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		<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>NBM</td>
<td>medium</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>Neural Basal Media is obtained by Invitrogen under their Gibco line of cell culture reagents.</td>
</tr>
<tr>
<td>E18 rat embryos</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Obtained from pregnant Sprague Dawley Rats. The pregnant rat is sacrificed and the E18 fetal rat pups dissected to obtain the E18 fetal rat cortex.</td>
</tr>
<tr>
<td>CMFM dissection buffer HBSS</td>
<td>buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>ice-cold Calcium-free Magnesium-free dissection buffer HBSS based.</td>
</tr>
<tr>
<td>15 ml Tube</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>Falcon No. 352097</td>
<td>Available through Fisher Scientific catalog number 14-959-70C</td>
</tr>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>37&#x02DA;C water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipets</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>glass</td>
</tr>
<tr>
<td>DMEM</td>
<td>medium</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>serum, used at 10% in DMEM</td>
</tr>
<tr>
<td>centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>set at 2500 rpm </td>
</tr>
<tr>
<td>Trypan Blue</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>for counting live/dead cells</td>
</tr>
<tr>
<td>BD Falcon Cell Strainer 40 um</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>Falcon cat# 352340</td>
<td>Available through Fisher Scientific. Fisher catalog# 08-771-1</td>
</tr>
<tr>
<td>B27</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>B27 is a proprietary supplement available through Invitrogen under their Gibco line of cell culture reagents.</td>
</tr>
<tr>
<td>Glutamax</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>35050-061</td>
<td>Glutamax is available through Invitrogen under their Gibco line of celll culture reagents.</td>
</tr>		</tbody>
		</table>
		</div>

preparing e18 cortical rat neurons for compartmentalization in a microfluidic device

Dans cette vidéo, nous démontrons la préparation de la E18 neurones de rat corticale. E18 neurones corticaux de rat sont obtenus à partir de cortex de rat fœtal E18 précédemment disséqué et préparé. Le cortex E18 est, à la dissection, immédiatement dissocié en neurones individuels. Il est possible de stocker E18 cortex dans Hibernate tampon E contenant B27 à 4 ° C jusqu&#39;à une semaine avant la dissociation est réalisée. Cependant, il y aura une baisse de la viabilité cellulaire. Généralement, nous obtenons de nos frais E18 Cortex. Il est transporté au laboratoire dans la glace de calcium libre tampon froid de dissection magnésium libre (CMFM). À l&#39;arrivée, la trypsine est ajoutée au cortex à une concentration finale de 0,125%. Le cortex est ensuite incubée à 37 ° C pendant 8 minutes. FBS DMEM contenant 10% est ajouté au cortex pour arrêter la réaction. Le cortex est ensuite centrifugé à 2500 rpm pendant 2 minutes. Le surnageant est éliminé et 2 ml de milieux de base neurale (NBM) contenant 2% de B27 (vol / vol) et 0,25% Glutamax (vol / vol) est ajouté au cortex, qui est ensuite remis en suspension par aspiration et refoulement. Ensuite, le cortex est trituré avec des pipettes en verre poli au feu précédemment, chacun avec une petite ouverture successive. Après trituration, le cortex est de nouveau centrifugé à 2500 rpm pendant 2 minutes. Le surnageant est alors éliminé et le culot remis en suspension du cortex avec 2 ml de NBM contenant B27 et Glutamax. La suspension cellulaire est ensuite passé à travers un tamis en nylon 40 cellules um. Suivant les cellules sont comptées. Les neurones sont maintenant prêts à charger dans l&#39;appareil neurone microfluidique.

Watch this Scientific Journal Video about Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device at JoVE.com

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

Dans cette vidéo, nous démontrons la préparation de la E18 neurones de rat corticale.

Préparation E18 neurones de rat corticale compartimentation dans un dispositif microfluidique

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

preparing-e18-cortical-rat-neurons-for-compartmentalization

Research

JoVE Journal

Biology

19.6K vues.  University of California, Irvine (UCI). Dans cette vidéo, nous démontrons la préparation de la E18 neurones de rat corticale.

Vidéo: Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Culture organotypique des cerveaux de rat E18

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Recherche

Biologie

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabrication d&#39;un dispositif microfluidique pour la compartimentation du Neuron Soma et des axones

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

Un dispositif microfluidique avec des modèles Groove pour étudier le comportement cellulaire

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Un dispositif microfluidique Dégradé génératrices de biologie cellulaire

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

T CD4 + des lymphocytes capturer en utilisant une puce jetable microfluidiques pour le VIH

Cell Capture Using a Microfluidic Device

Capturez cellule à l&#39;aide d&#39;un dispositif microfluidique

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

Préparer T facteur de croissance cellulaire à partir de splénocytes de rat

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Évaluer la motilité Neural Stem Cell utilisant un gel d&#39;agarose à base dispositif microfluidique

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

Protocole pour les neurones sympathiques de rat Culture ganglions cervicaux supérieur (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

Imagerie calcique des neurones corticaux utilisant Fura-2 heures

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...