Preparación de las neuronas de ratas fetales E18 cortical de compartimentación Antes de salir, es importante para calentar todos los medios necesarios y los reactivos a 37 ° C. También es importante esterilizar todo lo que se utiliza para la preparación de las células (por ejemplo, los bulbos de goma, bastidores de tubos, botellas de los medios de comunicación, etc), y lo que se coloca en la capilla, al lavarlo con etanol al 70%. <ol><li> Coloque dos trozos de corteza de rata fetal E18 (un cerebro, previamente disecados) en un tubo de 15 ml que contiene 1 ml de helado de calcio sin magnesio libre de tampón de disección. </li><li> Añadir 1 ml de 0,25% de tripsina-EDTA a la corteza en un tampón de disección, con lo que el volumen final de 2 ml y la concentración final de tripsina al 0,125%. </li><li> Coloque el tubo de 15 ml en un baño de agua 37 º C durante 8 minutos. </li><li> Durante este tiempo, el fuego de uñas 3 pipetas Pasteur de vidrio, formando aberturas cada vez más pequeños, en un gabinete de bioseguridad para ayudar a mantener la esterilidad. </li><li> Después de la incubación de 8 minutos de la corteza, agregar 10 ml de DMEM con FBS al 10% de la corteza cerebral para ayudar a detener la reacción de la tripsina. </li><li> Centrifugar el tubo de 15 ml que contiene la corteza y DMEM/10% FBS a 2500 rpm durante 2 minutos. </li><li> En la cabina de bioseguridad, separar el sobrenadante de la corteza con una pipeta Pasteur de vidrio con aspiración al vacío adjunto. Tenga cuidado de no perturbar o desalojar el pellet. </li><li> Añadir 1 ml de la NMB a la bolita corteza y suavemente la pipeta hacia arriba y abajo. Es muy importante evitar la creación de burbujas de aire, mientras que pipeta hacia arriba y abajo, como las burbujas de aire pueden dañar las células de la oxidación. </li><li> El uso del fuego pulido pipeta Pasteur con la más amplia apertura para triturar la corteza adjuntando una pera de goma estériles para el final y pipeta hacia arriba y abajo 5 veces, de nuevo teniendo cuidado de no introducir burbujas de aire. Continuar este proceso con la pipetas de vidrio otros dos, cada uno con un tamaño de abertura menor. </li><li> Después de la trituración, se centrifuga a las células de nuevo a 2500 rpm durante 2 minutos. </li><li> Después de la centrifugación, una vez más, separar el sobrenadante y resuspender el botón celular en 2 ml de NBM. </li><li> Filtrar la solución de células resuspendidas a través de un colador de la célula 45 um. </li><li> Tinción de las células con azul de tripano, se cuentan y se carga en los dispositivos. Por lo general carga de 20 ul de células por dispositivo. </li></ol> Nota: la concentración final de células es típicamente entre 2,5 millones de células / ml y las células de 8 millones / ml Carga de las células Después que las células han sido contadas, es el momento de la carga de las células en el dispositivo: <ol><li> Lleve los dispositivos previamente preparados que contienen NBM en la cabina de bioseguridad para mantener la esterilidad. </li><li> Quite el exceso de los pozos de succión al vacío. Sin embargo, tenga cuidado para evitar la eliminación de todos los medios de comunicación - que debe haber algunos medios de comunicación en el canal principal. </li><li> Aplicar 20 ul de células para el depósito superior izquierda del dispositivo (vea el diagrama si es necesario). Las células de flujo en el dispositivo y se adhieren a la superficie tratada PLL. </li><li> Después de la carga, coloque los dispositivos que contienen las células de nuevo en una incubadora durante 10 minutos para permitir que las células que desea adjuntar. </li><li> Después de 10 minutos, llenar los depósitos con los medios de comunicación y dispositivos de volver a meterla en la incubadora. </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>

Densidades celulares pueden variar dependiendo de la aplicación. Sin embargo, la siembra de las neuronas primarias de muy baja densidad de un general conduce a la muerte celular. Dependiendo de la incubadora y el nivel de humedad, los medios de comunicación puede ser necesario cambiar los dispositivos cada 2 ó 3 días. Al cambiar los medios de comunicación, es importante después de la eliminación de los medios de comunicación de los depósitos, a no quitar el medio de los canales principales. Además, es una buena práctica para poner los medios de comunicación fresca en los pozos de la parte superior y permitir que algunos medios de comunicación fresca que fluya a través del dispositivo y en los principales canales antes de llenar los embalses.

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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

En este video, se demuestra la preparación de las neuronas de ratas E18 cortical. E18 neuronas corticales de rata se obtienen de la corteza de ratas fetales E18 previamente disecados y preparados. La corteza es E18, la disección, de inmediato se disocian en las neuronas individuales. Es posible almacenar E18 corteza en Hibernate buffer E que contienen B27 a 4 ° C hasta una semana antes de la disociación se realiza. Sin embargo, habrá una disminución de la viabilidad celular. Normalmente obtenemos nuestro E18 corteza fresca. Se transporta al laboratorio en el hielo frío buffer de calcio libre disección de magnesio libre (CMFM). A su llegada, la tripsina se agrega a la corteza a una concentración final de 0,125%. La corteza se incuba a 37 ° C durante 8 minutos. DMEM con FBS al 10% se agrega a la corteza para detener la reacción. La corteza se centrifuga a 2500 rpm durante 2 minutos. Se elimina el sobrenadante y 2 ml de medio basal neural (NBM), que contiene 2% de B27 (vol / vol) y 0,25% Glutamax (vol / vol) se añade a la corteza que luego se volvió a suspender pipeteando arriba y hacia abajo. A continuación, la corteza se tritura con pipetas de vidrio previamente pulidas al fuego, cada uno con una pequeña apertura sucesiva. Después de trituración, la corteza es una vez más, se centrifuga a 2500 rpm durante 2 minutos. El sobrenadante se retira la corteza y el pellet resuspendido en 2 ml de NBM que contienen B27 y Glutamax. La suspensión celular se hace pasar por un colador de 40 um celular nylon. A continuación las células se cuentan. Las neuronas están listos para cargar en el dispositivo de microfluidos neurona.

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

En este video se demuestra la preparación de neuronas de rata E18 cortical.

Preparación de neuronas de rata E18 cortical de compartimentación en un dispositivo de microfluidos

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 vistas.  University of California, Irvine (UCI). En este video se demuestra la preparación de neuronas de rata E18 cortical.

Video: 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. 

Cultura Cortar organotípicos de los cerebros de ratas 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, ...

Investigación

Biología

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.

Fabricación de un dispositivo de microfluidos para la compartimentación de las neuronas y los axones Soma

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 dispositivo de microfluidos con los patrones de Surco para el estudio del comportamiento celular

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 dispositivo de microfluidos gradiente de generación de Biología Celular

 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

CD4 + T-linfocitos capturar usando un chip de microfluidos desechables para el VIH

Cell Capture Using a Microfluidic Device

Captura de células utilizando un dispositivo de microfluidos

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.

La preparación de Factor de Crecimiento Celular T a partir de esplenocitos de rata

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.

La evaluación de la motilidad de células madre neurales uso de un gel de agarosa al dispositivo basado en microfluídica

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.

Protocolo para las neuronas simpáticas de rata cultivo Ganglios cervical superior (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.

Imagen de calcio de las neuronas corticales con Fura-2 a.m.

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