Este trabajo fue apoyado por el NIH subvención NS27501 a DKOD. El apoyo adicional para este trabajo fue proporcionado por una beca de la Universidad de California Irvine en apoyo de DKOD a través del Programa profesor del HHMI.

Los preparativos antes del día de cultivo: <ol><li> Hacer una solución estéril de disección. </li><li> Hacer estéril DMEM y mantenerse en alícuotas de 10 ml a 4 º C durante 2 semanas. </li><li> Hacer estéril DDM2 suplementos y congelar en 50 o 100 alícuotas de 1 mes. </li><li> Hacer ConA / laminina. </li><li> Cubreobjetos de abrigo. Opcional: Haga CNBM estéril y guardar congelado hasta por 4 meses. </li></ol> En el día de cultivo I. Preparar la solución de la enzima (ES) en la campana de flujo laminar <ol><li> Poner 5 ml de solución de disección (DS) en un tubo de centrífuga de 15 ml. </li><li> Pesar 0,8 mg de L-cisteína en un eppendorf de 0,6 ml y añadir 150 ml de DS. Pipeta hacia arriba y abajo hasta que los cristales se han ido. </li><li> Añadir 150 ml de DS / cisteína al 5 DS ml y mezclar bien. </li><li> Añadir 50 U (unidades) de la papaína. </li><li> 7 ml de 0,1 N de Na OH y mezclar bien. Solución clara a los 30 minutos. cuando se activa. </li><li> Solución de la enzima de filtro con filtro de 0,2 mm en una jeringa estéril de 1,5 ml tubo de microcentrífuga tapa rosca Papaína: ¿Quieres 50 U en 5 ml DS. Cada lote es un poco diferente, por lo que debe calcular la cantidad de: 37,3 mg / ml y 28,3 U / mg 37,3 x 28,3 = 1055,59 U / ml (1000 ml) 50 U = 47,4 ml </li></ol> II. Hacer DDM2 medios En el día de cultivo: añadir los siguientes complementos para hacer DDM2 A 10 ml de DMEM añadir los suplementos, justo antes de cultivo: <ol><li> 100μl de transferrina </li><li> 100μl putrescina </li><li> 100μl selenio </li><li> La progesterona 100μl </li><li> La insulina 50μl </li><li> 10μl de 20 Hydroxyecdysone </li></ol> Nota: Asegúrese DDM2 suficiente para todas las culturas planificadas (cada cerebro colocadas en un portaobjetos simple en un individuo de 35 mm placa de Petri, 1,5 m de los medios de comunicación / cultura) Paso III-VI puede estar en una mesa de laboratorio hecho estéril y debe ser completado en 45 minutos o menos. III. Pupas Colección <ol><li> Seleccione 10-15 pupas de los lados del vial con un microscopio de disección. </li><li> Lugar pupas en la tapa de una seca de 35 mm placa de Petri. </li></ol> Nota: Por lo general, utilizan los cerebros de las pupas entre 55-78 horas después de la formación de pupa (APF). Es un poco más difícil de diseccionar el cerebro de las pupas jóvenes ya que las cápsulas cefálicas y tienden a colapsarse, mientras que el nivel general de crecimiento de las neuritas es ligeramente más baja desde el más antiguo pupas. En la cepa de tipo salvaje Cantón-S, estas etapas son reconocidos por los ojos pigmentados que son de color marrón claro a rojizo poco, con poco pigmento en otros lugares. IV. Decapitación bajo microscopio de disección <ol><li> Coloque dos gotas de solución estéril de disección en la tapa de la placa de Petri, junto a las pupas. </li><li> Aplique suavemente una sola pupa con unas pinzas finas en la mano izquierda y el uso de una aguja de calibre 27 jeringa conectada a una jeringa de 1 cc en la mano derecha para eliminar la cutícula en el extremo anterior de la pupa. </li><li> Apriete pupa poco con pinzas, y como cabeza emerge uso aguja de calibre 27 para cortar el cuello. </li><li> Con la punta de la aguja o con pinzas, la transferencia de la cabeza a la caída de la disección de una solución. </li><li> Repita hasta que haya 10-15 cabezas en una sola gota. </li></ol> V. Eliminación del cerebro de la cabeza bajo el microscopio de disección <ol><li> En cada mano, mantener una jeringa de 1 cc con una aguja de calibre 27. </li><li> Utilice estos a la posición de la cabeza de mosca en la caída de la disección de una solución para la probóscide es hacia arriba y la superficie dorsal de la cabeza es el norte. </li><li> Inserte la aguja a la izquierda en la trompa de echar la cabeza hacia abajo y el uso de la aguja derecha para hacer una raja en el ojo derecho, que va desde el ventral a la superficie dorsal. </li><li> Insertar la aguja justo al lado de la izquierda en la región de la trompa y luego usar la aguja izquierda para presionar suavemente la cutícula sobre el ojo izquierdo, empujando el cerebro hacia el corte en el ojo derecho. El cerebro debe surgir de la ranura de la derecha, relativamente intacto, a menudo con ambos he ópticabes aún está unido y en ocasiones el tejido ocular pigmentado así. </li><li> Empuje el cerebro en la parte posterior de la aguja y la transferencia de una gota de solución salina estéril de disección en un nuevo plato de estériles de 35 mm de Petri. </li><li> Recoger 10-15 cerebro para cada genotipo. </li></ol> VI. Eliminación de los lóbulos ópticos y tratamiento enzimático <ol><li> En cada mano, mantener una jeringa de 1 cc con una aguja de calibre 27 y usarlas para recopilar todos los cerebros en una pequeña área de la gota de la solución de disección </li><li> Use jeringas, herramientas de corte para eliminar los lóbulos ópticos de cada cerebro para el resto del tejido de la cultura es la región del cerebro. </li><li> Use un Pipetman 20 l, del 5 l, para transferir todos los cerebros de un solo genotipo a un tubo eppendorf con 1 ml de solución de papaína estéril. </li><li> Los cerebros se incubaron en la papaína durante 10-15 minutos en un conjunto rotatorio a 60 rpm. </li><li> Centrifugar a 3000 rpm durante 3 minutos. </li></ol> Dentro de campana estéril de flujo laminar - todas las medidas que el tejido es expuesto al aire se debe hacer en campana de flujo laminar para el resto de pasos. VII. Lavado <ol><li> Eliminar la solución de la enzima y reemplazar con una solución estéril de disección. </li><li> Centrifugar a 3000 rpm durante 3 minutos. </li><li> Lavar con una solución estéril de la disección de un total de 3 veces. </li><li> Quitar y reemplazar la disección de solución salina con DDM2. </li><li> Centrifugar y lavar con un total de 2 veces con DDM2. </li><li> La transferencia de los cerebros y los medios de comunicación a una estéril de 35 mm placa de Petri. </li></ol> VIII. Trituración y de las planchas bajo microscopio de disección <ol><li> Lugar 5 ul de DDM2 en el centro de un cubreobjetos. </li><li> La transferencia de un cerebro a esta caída de DDM2 con una punta amarilla. </li><li> Bajo el microscopio, el uso de agujas de calibre 27 dados hasta el cerebro en pedazos pequeños (se debe tomar &lt;1 minuto). </li><li> Bajo la guía visual, rompa la punta de una pipeta de vidrio que ha sido puesto a un punto fino para que las piezas más grandes pueden ser absorbidos por el cuidado extremo de la pipeta y expulsados ​​de nuevo en el medio. Usamos tubos de boca de succión que vienen de serie con 100 l de vidrio de hematocrito capilar. Nota: Tenga cuidado de no tener burbujas en los medios de comunicación y que tendrá que determinar empíricamente la pipeta de mejor tamaño para su uso. Los buenos le permiten disociar el cerebro con algunas células individuales y aún así algunos grandes grupos, en unos 30 segundos / cerebro. Cuando se mira a estos a una potencia mayor, aproximadamente el 10-20% de las neuronas todavía debe tener algunos procesos pendientes y todavía habrá algunos grandes grupos. Si se disocian demasiado, entonces todas las células se ronda y ellos han sufrido tanto daño que no va a sobrevivir. Este paso requiere práctica y debe hacerse rápidamente, ya que hay una gran superficie-a-volumen y la osmolalidad y el pH de la caída de DDM2 pueden cambiar rápidamente. Tejido tiene que ser colocado en incubadora de CO 2 tan pronto como sea posible. </li><li> Escribir en la tapa de la placa de Petri: &quot;genotipo, fecha y hora&quot;. </li><li> Coloque la placa de Petri de 35 mm en una placa de Petri de 100 mm (con Kimwipe húmedo) y dejar reposar en la incubadora de CO 2 durante 30 minutos. </li><li> Inundar el plato de 35 mm con 1,5 ml de DDM2. </li><li> Mantenga las culturas en la incubadora en un 5% incubadora de CO 2 (23 ° C). Nota: Si usted no tiene acceso a un enfriamiento incubadora de CO 2, utilizar un estándar de cultivo de tejidos de mamíferos incubadora y, o bien ponerlo en un cuarto frío y el calor a 23 ° C, o poner en el laboratorio, cierre temporal, y el uso de hielo en una bandeja en la parte inferior de la incubadora para regular temperatura ambiente alrededor. </li></ol> IX. Las células de alimentación <ol><li> El día 1 (día siguiente), añadir 0,5 ml de CNBM/B27 (Media acondicionado) para hacer DDM2 03:01: CNBM/B27. </li><li> El día 5 reemplazar 1 ml de los medios de 3:01 DDM2: CNBM/B27. </li><li> Mantener las células de alimentación con 3:01 DDM2: CNBM/B27 cada 4-5 días. Nota: Los cultivos pueden crecer sin los medios de comunicación acondicionado no neuronales, pero las neuronas parecen un poco más frágil y son más difíciles para su uso en experimentos de electrofisiología. </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1991).</li><li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+Establishing+and+Modifying+synaptic+circuits+in+the+Drosophila+Genetic+System.">Cellular bases of behavioral plasticity: Establishing and Modifying synaptic circuits in the Drosophila Genetic System.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+and+firing+properties+of+embryonic+Drosophila+neurons+grown+in+a+chemically+defined+medium.">Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.</a> J. Neurobiol. 27, 113-126 (1995).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+excitatory+synaptic+transmission+mediated+by+nAChR+in+cultured+Drosophila+neurons.">Fast excitatory synaptic transmission mediated by nAChR in cultured Drosophila neurons.</a> J. Neurosci. 19, 5311-5321 (1999).</li><li>Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+synaptic+currents+in+Drosophila+mushroom+body+Kenyon+cells+are+mediated+by+alpha-bungarotoxin-sensitive+nAChRs+and+picrotoxin-sensitive+GABA+receptors.">Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by alpha-bungarotoxin-sensitive nAChRs and picrotoxin-sensitive GABA receptors.</a> J. Neurosci. 23, 9246-9253 (2003).</li><li>Jiang, S. A., Campusano, J. M., Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+mushroom+body+Kenyon+cells+generate+spontaneous+calcium+transients+mediated+by+PLTX-sensitive+calcium+channels.">Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.</a> J. Neurophysiol. 94, 491-500 (2005).</li><li>Campusano, J. M., Su, H., Jiang, S. A., Sicaeros, B., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nAChR-mediated+calcium+responses+and+plasticity+in+Drosophila+Kenyon+cells.">nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells.</a> Develop Neurobiol. 67, 1520-1532 (2007).</li><li>Oh, H. -. W., Campusano, J. M., Hilgenberg, L. G. W., Sun, X., Smith, M. A., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+chemical+synapses+and+gap+junctions+between+Drosophila+brain+neurons+in+culture.">Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture.</a> Develop Neurobiol. , (2007).</li></ol>

Las neuronas cosecha de los cerebros de los roedores embrionarias / postnatal se puede cultivar en cultivo de células primarias, donde se extienden neuritas y la forma funcional de conexiones sinápticas. Métodos para la preparación de estas culturas están bien establecidos y los estudios en cultivos de neuronas de roedores han jugado un papel fundamental en la identificación de genes y factores ambientales implicados en la regulación de la formación de sinapsis y la función (Banker y Goslin, 1991). Mientras que las neuronas de inse...

Cubreobjetos de recubrimiento: Ponga en un autoclave cubreobjetos de 60 mm placa de Petri. Pipeta de 5 ul de Con una mezcla / laminina en el centro de cada cubreobjetos. Lugar en 37 ° C incubadora durante 2 horas. Enjuague cubreobjetos 3x con 100 ul de agua esterilizada cada uno. El uso de vacío conectada a una pipeta Pasteur estéril. Durante el último enjuague, levante el cubreobjetos con unas pinzas y seque ambos lados. Transferir el portaobjetos a una placa de Petri de 35 mm. Conservar a temperatura ambiente durante un máximo de un mes.

primary neuronal cultures from the brains of late stage drosophila pupae

En este vídeo, se demuestra la preparación de cultivos primarios de neuronas del cerebro de Drosophila finales de etapa de pupas. El procedimiento comienza con la eliminación de los cerebros de los animales en 70-78 horas después de la formación pupario. Los cerebros aislados se presentan después de la incubación breve en papaína seguido de varios lavados en medio de cultivo libre de suero. El proceso de separación mecánica de cada cerebro en una caída de 5 ul de los medios de comunicación en un cubreobjetos se ilustra. Los axones y las dendritas de las neuronas post-mitóticas son sheered cuando esté cerca del soma en la disociación, pero las neuronas comienzan a regenerarse los procesos dentro de unas horas de la siembra. Las imágenes muestran las culturas en vivo en 2 días. Las neuronas continúan elaborando los procesos durante la primera semana de la cultura. Poblaciones neuronales específicas, se pueden identificar en la cultura de uso de líneas GAL4 para dirigir la expresión específica de tejido de los marcadores fluorescentes, como las buenas prácticas agrarias o RFP. Grabaciones de células enteras han demostrado los cultivos de neuronas forman sinapsis funcionales, colinérgicas y GABAérgicas espontáneamente activo. Un segmento corto video ilustra la dinámica del calcio en las neuronas cultivadas con Fura-2 como un tinte indicador de calcio para controlar los transitorios espontánea de calcio y la nicotina respuestas evocadas de calcio en un plato de neuronas cultivadas. Estas culturas el cerebro de pupa son un modelo útil en el que las herramientas genéticas y farmacológicas se puede utilizar para identificar los factores intrínsecos y extrínsecos que influyen en la formación y función de las sinapsis centrales.

Watch this Scientific Journal Video about Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae at JoVE.com

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

Este video muestra la preparación de cultivos primarios de neuronas del cerebro de Drosophila pupas etapa tardía. Puntos de vista de las culturas show en vivo de crecimiento de las neuritas y la imagen de los niveles de calcio con 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 ...

primary-neuronal-cultures-from-brains-late-stage-drosophila

Research

JoVE Journal

Biology

15.0K vistas.  University of California, Irvine (UCI). Este video muestra la preparación de cultivos primarios de neuronas del cerebro de Drosophila pupas etapa tardía. Puntos de vista de las culturas show en vivo de crecimiento de las neuritas y la imagen de los niveles de calcio con Fura-2.

Video: Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

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 ...

Investigación

Biología

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Las culturas de las células nerviosas de Aplysia para imágenes de alta resolución de los Conos de Crecimiento

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria disociada Mesencéfalo culturas dopamina celulares de roedores recién nacidos

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

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

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

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

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

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

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 ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Preparación de desarrollo y de adultos Drosophila Los cerebros y retinas de imágenes en vivo

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

El establecimiento de cultivos primarios de fibroblastos de adultos de los roedores

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Cultivos de células primarias de Drosophila Gástrula embriones

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells Aislamiento de Primaria cultivos de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

La utilidad de la etapa específica-Mediados o finales<em&gt; Drosophila</em&gt; Aislamiento folículo

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...