Este estudio fue financiado en parte por los fondos iniciales de la Texas A &amp; M University

Preparación antes de la disección Instrumentos quirúrgicos: esterilizar todos tijeras y pinzas de disección por autoclave Cubreobjetos de limpieza: <ol><li> Ponga todos los cubreobjetos (15 mm de diámetro por 24 y placa) en el vaso 1 litro de hierba en el 33% de HCl por lo menos 24 h </li><li> Lavar con agua corriente durante 10 minutos con un movimiento de vez en cuando para eliminar todo residuo de HCl </li><li> Enjuague con agua Millipore </li><li> Drenar el agua </li><li> Remoje cubreobjetos en el 95-98% de etanol </li><li> Cubreobjetos traslado a un paño limpio sobre una placa plana y el aire seco del cubreobjetos </li><li> Transferencia cubreobjetos a un vaso de vidrio, cubrir con papel de aluminio y autoclave Nota: Los cubreobjetos se pueden almacenar en esta etapa </li><li> Coloque el cubreobjetos individuales en placas de cultivo. Listo para el revestimiento </li></ol> Cubreobjetos recubrimiento: <ol><li> Diluir 100 acciones x de poli-D-lisina (PDL, 10 mg / ml en el 0,5% de albúmina sérica bovina en PBS, almacenados en alícuotas a -20 ° C) con PBS y el filtro de esterilización (0,22 m) </li><li> Cubreobjetos cubrir con una solución x PDL (~ 0,15 ml / cm 2) durante 2 horas a 37 ° C en una incubadora </li><li> Retire la solución PDL y lavar 3 veces con agua estéril O ddH 2 y cubreobjetos se seque por completo </li></ol> Nota: El pelo lo suficientemente cubreobjetos / placas para cada disección. PDL recubierto cubreobjetos se puede almacenar durante un par de semanas a 4 ° C. Los medios de comunicación y soluciones Medio de la disección (DM): El uso de solución salina equilibrada estéril helada de Hank (w / o Ca 2 +, Mg 2 +) (HBSS, Invitrogen 14.175), complementado con HEPES 10 mM (Invitrogen 15630). Los medios de comunicación agregado cultural: El medio DMEM/NBB27 contiene DMEM / Neurobasal (1:1 vol: vol), el 2% B27, 1 x Sato, 0,5 mM piruvato sódico, 0,75 Glutamax, 60μg/ml N-acetilcisteína, 5 mg / ml la insulina, 10 nM d-biotina y el 1% penicilina / estreptomicina. Para hacer 100 ml del medio de cultivo total, mezclar 50 ml de DMEM (w / o piruvato / glutamina, Invitrogen 11.960), 50 ml de medio Neurobasal (Invitrogen 211.034), 375 l Glutamax (100 x, Invitrogen 35.050), 5,5 mg de sodio piruvato (Sigma P2256), 2 ml B27 (Invitrogen 17.504), 6,3 mg de N-acetilcisteína (Sigma A8199), 1 ml de Sato acciones (100 acciones x, ver más abajo), 25 l de d-biotina madre (4000 x acciones, 40 M en PBS almacenado en alícuotas a -20 ° C. Sigma B4639), 100 l de la insulina (1000 x stock, 5 mg / ml de HCl 0,01 N almacenado en alícuotas a -20 ° C, Sigma 16 634), y 1 ml de penicilina / estreptomicina (100 x acciones, Invitrogen 15.140), filtro de esterilizar y almacenar a 4 ° C. Sato 100 x solución de reserva: Para hacer 40 ml de la acción: mezcla de 40 ml Neurobasal con 400 mg apo-transferrina (Sigma T2252), 400 mg de BSA (Sigma A9647), 10 l de progesterona (25 mg / ml de etanol, se almacena como alícuotas a -20 ° C. Sigma P8783), 64 mg putrescina (Sigma P7505), y 40 l de selenito de sodio (30 mM en PBS, almacenados en alícuotas a -20 ° C, Sigma S5261). Filtro de esterilización de la solución madre de Sato, y almacenar en alícuotas a -20 ° C. Neurona medio chapado (PM): Media Neurobasal, el 2% B27, 2 mM de glutamina (stock 100x, alícuotas almacenadas a -20 ° C, Invitrogen 25.030), 25μM ácido glutámico (100 acciones, alícuotas almacenadas a -20 ° C) y el 1% penicilina / estreptomicina. Neurona medio de cultivo (CM): Media Neurobasal, el 2% B27, 2 mM de glutamina (stock 100x, alícuotas almacenadas a -20 ° C) y 1% de penicilina / estreptomicina. 5-FdU acciones: acciones 100x, 1mM 5-fluoro-2&#39;-desoxiuridina (Sigma F0503) and1mM uridina (Sigma U3003) en medio de Neurobasal. Filtro esterilizado y almacenado en alícuotas a -20 ° C. La papaína solución de digestión (hace fresco antes de la disección): <ol><li> Disolver 3,2 mg L-cisteína (Sigma C-7352) en 4 ml de DM </li><li> Ajustar el pH a aproximadamente 7,4 con NaOH 1 N (prueba de tiras reactivas de pH) y en un baño de agua a 37 ° C Nota: Realice el siguiente paso justo antes de la digestión del tejido (ver más abajo). </li><li> Añadir la papaína a una concentración final de 20 unidades / ml </li><li> Filtro de esterilización (0.22mm), y en un baño de agua a 37 ° C </li></ol> Tripsina inhibidor de la solución (que inmediatamente antes de su disección): <ol><li> Disolver 0,2 g de inhibidor de tripsina (Sigma T7295) en 20 ml DM </li><li> Verificar el pH y ajustar el pH a ~ 7,4 con NaOH 1 N </li><li> Filtro de esterilización, y en un baño de agua a 37 ° C </li></ol> La disección del cerebro Configuración: <ul><li> Pinzas esterilizadas y tijeras </li><li> 70% de etanol </li><li> Un pañuelo de papel limpio y DIAPer pad en el banco </li><li> Platos de Petri de 10 cm con helado de DM para el útero y el feto en el banco </li><li> De hielo en la plataforma microscopio de disección </li><li> 2-3 platos de 6 cm con DM en el hielo </li><li> PM caliente en un baño de agua 37 ° C </li></ul><ol><li> La eutanasia a una mujer embarazada de ratas Sprague-Dawley (E16) de acuerdo con un procedimiento aprobado por el Comité Institucional de nosotros Comité (IACUC) </li><li> Sentar las ratas en la plataforma de pañal y el alcohol de esterilización en la región abdominal </li><li> Use pinzas para sujetar la piel abdominal y un par de tijeras para hacer una incisión en forma de V corta solamente la piel </li><li> Separe la piel y el uso de otro pinzas esterilizadas y las tijeras para cortar a través de la capa muscular </li><li> Extracto de la cadena de los sacos embrionarios y transferirlos a un plato de 10 cm que contiene helada DM </li><li> Utilizando unas tijeras de microdisección, retire con cuidado cada embrión del saco y el cerebro de cada embrión, y el lugar liberado cerebro en un plato de 10cm limpio con DM en una plataforma de hielo </li><li> En una campana de laminar y bajo un microscopio de disección, retirar el cerebro medio / rombencéfalo secciones, y el uso de unas pinzas finas para eliminar las meninges, y la transferencia de la corteza limpia / hipocampo de un nuevo plato con 6 cm de DM en una bolsa de hielo Nota: en esta etapa, el tejido está listo para su digestión por enzimas. </li><li> Hacer la papaína y la solución de inhibidor de la tripsina y el filtro de esterilización </li><li> Eliminar el exceso de DM de corteza / hipocampo </li><li> Agregar 4 ml de la solución de papaína digestión preparado </li><li> La transferencia de todo el contenido de un tubo de 50 ml y colocar el tubo Falcon en un baño de agua a 37 ° C durante exactamente 5 minutos </li><li> Retire la solución con una pipeta de la papaína </li><li> Añadir 5 ml de solución de inhibidor de la tripsina, agitar el tubo, y colocar en un baño de agua a 37 ° C durante 2-3 minutos </li><li> Eliminar la solución de inhibidor de tripsina </li><li> Repita el paso 13 y 14 tres veces </li><li> Añadir PM caliente 20ml </li><li> Triturar hasta que todos los grupos han desaparecido (~ 20 a 30 veces) </li><li> Centrifugar a 100xg de 7min </li><li> Resuspender el precipitado en 10 ml de PM y lavar dos veces por centrifugación </li><li> Resuspender el precipitado en 10 ml de cultivos de neuronas PM regular o en NBB27/DMEM de las culturas agregado </li><li> Pasar a las células a través de un tamiz de células 70μm </li><li> Contador de células vivas </li><li> Proceder a la cultura global, como se describe a continuación o la placa de las células de cultivos de neuronas regulares Nota: En este paso, altamente enriquecido cultivos de neuronas se puede lograr mediante siembra de las células disociadas en las placas de recubrimiento PDL-con una densidad de 200 a 640 células / mm 2 en función de la finalidad de los experimentos. Después las células se han conectado (~ 2-4h), reemplace medio con PM caliente. Si el cultivo durante períodos prolongados de tiempo, el día 4, reemplace la mitad de la media con CM caliente. Para altamente purificada cultivos neuronales, inhibidor de la mitosis 5 FdU (concentración final 10 mM) se puede añadir en DIV2 para inhibir la no proliferación de las células neuronales (es decir, durante DIV2-3). Después de la recuperación en la CM durante 2 días, las células son el pulso de nuevo al tratamiento con 5-FdU durante 2 días (DIV 6-7) seguido de un cambio de medio completo. A partir de entonces, reemplazar la mitad de la media con CM dulce, caliente cada 3-4 días. Las neuronas son viables hasta por 4 semanas. </li></ol> Agregados de preparación y de las planchas <ol><li> Suspender las células disociadas en el medio que contiene 1x NBB27/DMEM Sato, CNTF 10ng/ml y forskolin 10μM a una densidad de 2x10 6 células / ml </li><li> Transferencia de 2 ml de suspensión celular en cada pocillo de un sin recubrimiento de 6 pocillos </li><li> Cultura por 3 noches. Cada día, suavemente volver a suspender las células una vez con un Pipetman P1000 Nota: Las células comienzan a formar agregados (Figura 1). </li><li> En el día de las placas, agregado, la capa de placa / cubreobjetos con Matrigel: <ul><li> Diluir acciones Matrigel (factor de crecimiento reducido, BD Biosciences 354230) 1:20 con DMEM frío en hielo Nota: alícuotas de Matrigel debe ser preparado por el protocolo del fabricante. Todas las puntas, tubos Eppendorf y soluciones para la toma de alícuotas de las acciones debe estar fría para evitar la polimerización Matrigel. Alícuotas de Matrigel se mantienen a -80 ° C y se descongelaron a 4 ° C. </li><li> Escudo previamente PDL-cubreobjetos recubiertos con 300μl/well de la solución diluida Matrigel durante la noche en una incubadora a 37 ° C </li><li> Lavar con PBS caliente, lavar la placa con agua tibia estéril O ddH 2 y dejar las placas en la incubadora. </li></ul></li><li> 3 días después de la formación de agregados, resuspender suavemente las células de nuevo y se tamiza la suspensión a través de una malla de 200μm en un tubo de 50 ml Falcon. Permita que los agregados se depositen en el fondo del tubo por la gravedad (~ 3-5min) </li><li> Retire con cuidado el sobrenadante </li><li> Añadir medio, suavemente volver a suspender los agregados y dejar a establecerse de nuevo. Repita este procedimiento varias veces para eliminar las células muertas, las células individuales y los escombros. Use el microscopio para verificar si el sobrenadante aún contienen las células no agregados y basuras </li><li> Resuspenda suavemente los agregados en el medio NBB27/DMEM mismo (Figura 1) y el recuento de los agregados </li><li> Ajustar la densidad de los agregados de alrededor de 25-30 aggreggats/50ul </li><li> Transferencia de alícuotas (500-1000μl) de la suspensión total de 2 ml tubos Eppendorf para la siembra Nota: Dado que los agregados tienden a asentarse en el fondo de los tubos, es crucial para hacer tubos múltiples de la suspensión total para la siembra. Resuspenda suavemente los agregados a menudo antes de la siembra que en cubreobjetos recubiertos o placas de cultivo para garantizar un número similar de agregados chapada en cada portaobjetos. </li><li> Saque la placa de cultivo que contienen Matrigel recubierto cubreobjetos de la incubadora. Retire la solución de cada pocillo, lavar los cubreobjetos con PBS caliente y se ddH2O, y luego brevemente en seco. Ahora están listos para la siembra de agregado </li><li> Invertir un tubo que contiene la suspensión total para que puedan ser distribuidos de manera uniforme antes de la siembra. Carga 50μl de la suspensión total en el centro de cada cubreobjetos, y colocó suavemente la placa posterior a la incubadora sin ningún tipo de perturbación adicional para permitir que los agregados para fijar de manera uniforme para el cubreobjetos Nota: La densidad de los agregados es crucial. Si los agregados se siembran demasiado cerca uno del otro o si la densidad de siembra es alta, tienden a fusionarse a medida que crecen. </li><li> Añadir DMEM/NBB27medium adicionales (500 l) a cada pocillo 4-6 horas más tarde o temprano la mañana siguiente </li><li> Para mantener la cultura global, la mitad de cambiar el medio cada 3-4 días. Cuando cambio de medio, tenga cuidado de no romper los agregados y esto se hace añadiendo lentamente un medio a lo largo de la pared lateral del pozo. Nota: Pocas horas después de agregados unida a la placa, crecimiento de las neuritas de los agregados se pueden observar. Los axones crecen rápidamente en las primeras dos semanas y forman abundantes conexiones entre los agregados (Figura 2). Gliales progenitores migran radialmente hacia fuera de los agregados y se diferencian en el tiempo en los astrocitos y oligodendrocitos maduros. </li></ol><img src="/files/ftp_upload/1304/1304fig1.jpg" alt="la figura 1" /> Figura 1. Imágenes de contraste de fase de células que forman los agregados en diferentes días y agregados después del lavado. <img src="/files/ftp_upload/1304/1304fig2.jpg" alt="la figura 2" /> Figura 2. Imágenes de contraste de fase de una cultura global 2 y 12 días después de sembradas en Matrigel recubierto cubreobjetos.

<ol>
	<li>Matthieu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+in+rat+brain+aggregating+cell+cultures.">Myelination in rat brain aggregating cell cultures.</a> Neuroscience. 3, 565-56 (1978).</li><li>Chen, Y. i. n. g. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat. Protocols. 2, 1044-10 (2007).</li><li>Watkins, T. A., Emery, B., Mulinyawe, S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system.">Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.</a> Neuron. 60, 555-55 (2008).</li><li>Svenningsen, A. F., Shan, W. e. i. -. S. o. n. g., Colman, D. a. v. i. d. R., Pedraza, L. i. l. i. a. n. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+method+for+culturing+embryonic+neuron-glial+cell+cocultures.">Rapid method for culturing embryonic neuron-glial cell cocultures.</a> Journal of Neuroscience Research. 72, 565-56 (2003).</li></ol>

Los primeros estudios informó la formación de sinapsis y de la mielina madura libre mediada la rotación de cultivos de agregados flotantes 1. El agregado del SNC sistema de cultivo se describe aquí se combina el suero libre de crecimiento de las células progenitoras multipotentes en tres dimensiones con los agregados de la comodidad de las culturas tradicionales en 2D para facilitar el análisis de desarrollo de las células, la migración y la diferenciación in vitro. El sistema puede ser modi...

preparation of rat brain aggregate cultures for neuron and glia development studies

Un sistema in vitro que recapitula el desarrollo y diferenciación de células progenitoras en neuronas maduras y células gliales en el sistema nervioso central (SNC) que proporcionan una poderosa plataforma para los neurocientíficos para investigar axo-glial interacciones, propiedades y la diferenciación de células progenitoras multipotentes, y la progresión de oligodendroglial células de linaje en el nivel celular y molecular. Se describe aquí un total de SNC sistema de cultivo de prosencéfalos embrionario de la rata, que puede ser mantenido en un medio libre de suero hasta 3-4 semanas y se utiliza en nuestro laboratorio como un modelo para estudiar las neuronas-glia la interacción y la mielinización del SNC. Este video clip se mostrará cómo aislar y cultivar estas culturas agregada del SNC de cerebro de rata E16. Además, a partir de la disección del cerebro mismo, altamente enriquecido regulares cultivos de neuronas disociadas se pueden obtener fácilmente y se utiliza para diversos estudios sobre las neuronas del SNC o utilizados para co-cultivos con otras células.

Watch this Scientific Journal Video about Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies at JoVE.com

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

A los protocolos de un sistema de cerebro de rata embrionaria cultura global se describe. Progenitoras multipotentes en los agregados pueden desarrollarse y diferenciarse en neuronas, astrocitos y oligodendrocitos.

Preparación de las Culturas Rata total del cerebro de las neuronas y la glía de Estudios para el Desarrollo

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

preparation-rat-brain-aggregate-cultures-for-neuron-glia-development

Research

JoVE Journal

Biology

12.3K vistas.  Texas A&M University (TAMU). A los protocolos de un sistema de cerebro de rata embrionaria cultura global se describe. Progenitoras multipotentes en los agregados pueden desarrollarse y diferenciarse en neuronas, astrocitos y oligodendrocitos.

Video: Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

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.

Cultivos neuronales primarios del cerebro de Drosophila pupas etapa tardía

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Investigación

Biología

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Preparación de cultivos neuronales a partir de embriones de Drosophila Midgastrula Etapa

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

ALS - Motor Neuron Disease: Mechanism and Development of New Therapies

Jeffrey D. Rothstein speaks about the pathology and mechanisms underlying amyotrophic lateral sclerosis or ALS, advances in ALS research, and current strategies towards the development of therapies.

ALS - enfermedad de la motoneurona: Mecanismo y desarrollo de nuevas terapias

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 ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

Preparación de los cultivos disociados ratón neuronas corticales

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

Preparación de 2-dGuo cultivos tratados con órganos timo

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Preparación de Aplysia Sensorio-motor culturas de células neuronales

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Un compartimiento de Multi-CNS neuronas-glia Co-cultivo de microfluidos Plataforma

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

La toxina de inducción y extracción de proteínas a partir de Fusarium Spp. Los cultivos para los estudios proteómicos

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Preparación de los tendones Cola de Rata de Estudios biomecánicos y Mechanobiological

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...