Nos gustaría dar las gracias a Ryan Maneri (Producciones ostrero, LLC) para filmar nuestro procedimiento y Rodney McPhail (Departamento de Ciencias Biológicas, Universidad de Purdue) para obtener ayuda con la edición de vídeo disección. La investigación en el laboratorio de Suter es apoyado por subvenciones del NIH (R01 NS049233) y el Centro Bindley Bioscience en la Universidad Purdue de DMS

Soluciones <ul><li> L15-ASW medio de cultivo celular (1l) <ul><li> 1 bolsa de polvo L15 </li><li> Añadir 800 ml de H 2 O ultrapura </li><li> NaCl 400 mM </li><li> MgSO4 27 mM </li><li> MgCl2 28 mM </li><li> L-glutamina 4 mM </li><li> Gentamicina 50 mg / ml </li><li> HEPES 5 mM </li><li> Ajustar el pH a 7.9 </li><li> Añadir gota a gota, CaCl2 9,3 mM (parada si se precipita) </li><li> Añadir H 2 O ultrapura a 1 l </li><li> Compruebe la osmolaridad (950-1000 mg / kg) con osmómetro (presión de vapor, Wescor # 5520) </li><li> Filtro de 0,22 micras utilizando una unidad de filtración a presión positiva (Filtros: SVGV010RS Millipore) </li><li> Almacenar a 4 ° C durante un máximo de un mes </li></ul></li><li> Poli-L-lisina (70-150 kD) 200 mg / ml de solución estéril de valores de H 2 O ultrapura se mantiene a -20 º C </li><li> 0,5 M de MgCl2 solución </li></ul> Día 1: La disección del ganglio abdominal de Aplysia Preparación de la solución enzimática disociación <ol><li> Pesar 10.9 mg de Dispasa enzima (Worthington, Cat: LS02111) en un tubo Eppendorf </li><li> Añadir 900 l de L15-ASW medio de cultivo, y 100 l de H 2 O estéril ultrapura </li><li> Mezcle y ponga a temperatura ambiente (TA) </li></ol> La disección del ganglio abdominal <ol><li> Llene una jeringa de 50 ml con 0,5 M de MgCl2 solución. </li><li> Tener un animal en el tanque, manejar con cuidado para evitar la respuesta animal entintado defensa. </li><li> Coloque la Aplysia a su lado en una tabla de disección (espuma de poliestireno bordo), parte rostral en el lado derecho, caudal a la izquierda. </li><li> Insertar la aguja en los animales justo detrás de la cabeza e inyecte toda la solución de MgCl 2 en la cavidad corporal. </li><li> Frote suavemente el animal para dispersar a la solución de MgCl 2 en toda la cavidad del cuerpo, espere unos minutos para que el animal anestesiado por completo. </li><li> Pin de la cabeza y la cola del animal a la mesa de disección con dos agujas. </li><li> El uso de fórceps para levantar la piel, cortar la piel y la capa muscular con unas tijeras de gran tamaño en la cabeza y la cola para hacer una gran abertura en el costado. </li><li> Encontrar el ganglio abdominal en el ovario similares a un cepillo. El uso de fórceps para mantener los nervios que conectan alrededor de 1 cm en la parte delantera de los ganglios abdominales, cortar los nervios con tijeras de disección en la parte delantera de la posición de pinzas de sujeción y por detrás del ganglio abdominal. </li><li> Ganglio lugar en la solución Dispasa e incubar durante 15-16 horas a 22 ° C utilizando un baño de agua con temperatura controlada. </li></ol> Día 2: Revestimiento de las células de la bolsa Aplysia La preparación de poli-lisina cubreobjetos <ol><li> En un banco de flujo, preparar tres platos de Petri de 35 mm como platos de trabajo para la disección de los ganglios, llenándolos de 4 ml L15-ASW medio cada uno. Transferir el ganglio abdominal de la solución dispasa a uno de los platos después de 15,5 horas de trabajo de la digestión. </li><li> Con unas pinzas tomar ácido limpiado # 1.5 cubreobjetos (22x22 mm) almacenados en etanol, eliminar el alcohol y esterilizar cubreobjetos por llamas brevemente sobre mechero Bunsen. Deje enfriar brevemente antes de colocar el cubreobjetos en las placas de cultivo. </li><li> Prepare una punta de placas curvas doblando una punta amarilla sobre el quemador Bunsen (llama brevemente para evitar la fusión de plástico!). </li><li> Preparar una cantidad apropiada de 20 mg / ml de solución de poli-lisina mediante la dilución de 200 mg / ml de solución madre con agua estéril H 2 O ultrapura. </li><li> Cubra cada portaobjetos con 500 l de 20 mg / ml de poli-lisina y se incuba durante 20 minutos a temperatura ambiente. </li><li> Lave cada cubreobjetos 3-5 veces con 500 l estéril H 2 O ultrapura cada uno. </li><li> Eliminar el agua de cubreobjetos, llenar cada plato con 4 ml de medio de cultivo L15-ASW. </li></ol> La disociación de las células de la bolsa <ol><li> Bajo un microscopio de disección, corte el ganglio abdominal en la mitad con tijeras de disección y pinzas previamente lavado con etanol al 95% (Figura 1 línea 1). </li><li> En una mitad del ganglio, corte entre el grupo bolsa de células y las células hemiganglion (Figura 1 línea 2), y cortar resto de las extensiones de los nervios en el otro lado de la bolsa de las células (Figura 1 línea 3). Trabajo en una mitad del ganglio en un momento, dejar la otra mitad en el plato, mientras que las células del primer grupo están chapados. </li><li> El uso de dos pinzas (o un fórceps y tijeras de disección), bolsa de lanzamiento de racimo de células, empujando a un lado de la vaina de tejido conectivo que rodea el cúmulo de células bolsa. Cortar el tejido conectivo con tijeras de disección, si es necesario. Tenga cuidado de no apretar y tocar el grupo de células bolsa demasiado durante este proceso. </li><li> Cuando la mayoría del tejido conectivo se elimina, la transferencia de la bag grupo de células en un plato nuevo trabajo con la punta amarilla doblada preparado con anterioridad. Tenga cuidado de no dejar que el grupo de células bolsa de quedar atrapado dentro de la punta de color amarillo o pegado en la interfase aire / medio plazo. </li><li> Triturar el grupo con la punta amarilla para eliminar las células individuales de bolsa. Para ello, pipetear el grupo dentro y fuera de la punta. Comience con bajas fuerzas de corte y aumentar la fuerza cuando sea necesario. Utilice siempre la fuerza mínima necesaria para remover las neuronas sin matar demasiadas células. </li><li> Recoger 3-5 células bolsa sana y transferirlos a un plato con un cubreobjetos poli-lisina. Evitar la captación de las células muertas (que son de color negro / transparente en el centro) y restos de tejido conectivo. </li><li> Repita el paso 6. y el lugar alrededor de 15-25 neuronas vivas en el centro de cada cubreobjetos. Si las células muertas o restos son trasladados, así, eliminar el material no deseado recogerlo o soplando fuera de las células sanas. Este proceso toma alrededor de 2.3 horas para ambos grupos. </li><li> Una vez finalizado mantener las placas de cultivo en el banco de flujo de por lo menos 2 horas a temperatura ambiente para permitir que las células se adhieren (mantener banco de flujo del ventilador apagado para evitar que las vibraciones no deseadas). </li><li> Colocar las cápsulas en una incubadora a 14 ° C durante la noche, o hasta su posterior utilización. Los conos de crecimiento se puede desarrollar en 4.10 horas. </li></ol><img src="/files/ftp_upload/old/662_2008_2_10_0/image001.jpg" alt="Figura 1. Esquemática de Aplysia californica ganglio abdominal. Líneas de color indican dónde cortar para obtener clusters bolsa de la célula." /> Figura 1. Esquemática de Aplysia californica ganglio abdominal. Líneas de color indican dónde cortar para obtener clusters bolsa de la célula. Día 3: Configuración de cámara para imágenes en vivo de las células de la bolsa Aplysia Montaje de imágenes de cámara <ol><li> Antes de utilizar las células de la bolsa de experimentos con células en vivo de imágenes, inspeccionar las células en un microscopio de baja potencia. Si las células no se han desarrollado los conos de crecimiento sano, el suministro de un nuevo medio y dejar a temperatura ambiente durante 1-2 horas. </li><li> Para suministrar las placas con medio fresco L15-ASW, eliminar la mitad (~ 2 ml) de medio de edad, con una línea de vacío, y añadir 2 ml de nuevo L15-ASW medio por placa de Petri. Es importante no eliminar por completo todo el medio del plato a la vez, ya que se eliminan las células del cubreobjetos. </li><li> Para obtener imágenes de bolsa de la célula, nos reunimos dos cubreobjetos y dos separadores de plástico en una cámara de imágenes de sándwich. El cubreobjetos encima del pastel se corta más corta para permitir un fácil intercambio de medios y reactivos. Durante el montaje, cubreobjetos con células bolsa adherida debe ser sumergido en el L15-ASW medio en todo momento para evitar que las células se desprenda. </li><li> Para preparar la cámara, cortar ~ 2 mm de lado derecho de un mm # 1 cubreobjetos 22x22 con diamante pluma (cortador de vidrio) y la regla. </li><li> Con una jeringa de 1 ml, aplicar grasa de alto vacío a ambos lados de la cubreobjetos cortos, y colocar separadores de plástico a cada lado. Aplicar grasa de vacío en la parte superior de los dos separadores. </li><li> Invertir el cubreobjetos y montaje espaciador, y ponerlo en un plato de Petri que contiene las células de la bolsa en un cubreobjetos. Alinear y presionar suavemente hacia abajo para pegar cubreobjetos arriba a abajo cubreobjetos con la punta opuesta de dos Q-tips. </li><li> Con unas pinzas, suavemente levante el conjunto del sándwich de la placa de Petri sin el medio de escape. Alinear los dos cubreobjetos, si es necesario, y limpie el exceso con un medio Kimwipe. Coloque el sándwich en la parte inferior del soporte portaobjetos y coloque la tapa con cuidado utilizando clips. </li><li> Limpiar el centro de la parte superior del sándwich con Q-tips se secó en el limpiador de cristales (Sparkle es utilizado como limpiador de cristales). Rápidamente secar el limpiador de cristales con un segundo y seco Q-tip para evitar las líneas consecutivas. Haga lo mismo con el cubre parte inferior. Cubreobjetos limpio, libre de líneas de racha son importantes para imágenes de alta calidad. </li></ol> Bolsa de células en el microscopio Desde Aplysia cultivos de células neuronales en la bolsa de baja densidad, lo mejor es que la configuración de su microscopio tiene funciones etapa de posicionamiento para facilitar el cambio entre las células. Las células de la bolsa puede ser fácilmente mantenido a temperatura ambiente durante varias horas en el escenario durante los experimentos de imagen. Cambio del medio periódicamente para evitar la evaporación.

<ol>
	<li>Forscher, P., Kaczmarek, L. K., Buchanan, J. A., Stephen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+induces+changes+in+distribution+and+transport+of+organelles+within+growth+cones+of+Aplysia+bag+cell+neurons.">Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons.</a> J. Neurosci. 7, 3600-3611 (1987).</li><li>Suter, D. M., Errante, L. D., Belotserkovsky, V., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ig+superfamily+cell+adhesion+molecule,+apCAM,+mediates+growth+cone+steering+by+substrate-cytoskeletal+coupling.">The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling.</a> J. Cell. Biol. 141, 227-240 (1998).</li><li>Schaefer, A. W., Kabir, N., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+and+actin+arcs+guide+the+assembly+and+transport+of+two+populations+of+microtubules+with+unique+dynamic+parameters+in+neuronal+growth+cones.">Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones.</a> J. Cell. Biol. 158, 139-152 (2002).</li><li>Suter, D. M., Schaefer, A. W., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+are+necessary+for+SRC+family+kinase-dependent+growth+cone+steering.">Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering.</a> Curr. Biol. 14, 1194-1199 (2004).</li></ol>

Aplysia neuronas de células bolsa de proporcionar un suero libre de cultivos de células neuronales del sistema con muy pocas células no neuronales. Estas neuronas forman los conos de crecimiento muy grande adecuado para hacer frente a una serie de preguntas de biología celular importante. Bolsa de neuronas de células pueden ser instrumentalizadas fácilmente y con imágenes a temperatura ambiente durante varias horas. Utilizando microscopía de fluorescencia moteado (FSM), una cuantitativa puede analizar los distintos parámetros de F-...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>#1 glass coverslips 22x22 mm</td>
<td>Tool</td>
<td>VWR</td>
<td>48366067</td>
<td>&nbsp;</td>
<tr>
<td>#1.5 glass coverslips 22x22 mm</td>
<td>Tool</td>
<td>Corning</td>
<td>2870-22</td>
<td>&nbsp;</td>
<tr>
<td>35 mm Petri dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Filters for medium filtration</td>
<td>Tool</td>
<td>Millipore</td>
<td>SVGV010RS</td>
<td>&nbsp;</td>
<tr>
<td>High vacuum grease</td>
<td>Tool</td>
<td>Dow Corning</td>
<td>1597418</td>
<td>&nbsp;</td>
<tr>
<td>Osmometer</td>
<td>Tool</td>
<td>Wescor</td>
<td>5520</td>
<td>&nbsp;</td>
<tr>
<td>Plastic shims</td>
<td>Tool</td>
<td>Small Parts Inc </td>
<td>SHSP-200</td>
<td>&nbsp;</td>
<tr>
<td>Calcium chloride</td>
<td>Reagent</td>
<td>Fisher</td>
<td>C79-500</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15750-060</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H4030</td>
<td>&nbsp;</td>
<tr>
<td>L15 medium</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>41300-039</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G8540</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride</td>
<td>Reagent</td>
<td>Mallinckrodt</td>
<td>5958-04</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium sulfate</td>
<td>Reagent</td>
<td>Mallinckrodt</td>
<td>6070-12</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-lysine (70-150 kD) </td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6282</td>
<td>&nbsp;</td>
<tr>
<td>Sodium chloride</td>
<td>Reagent</td>
<td>Mallinckrodt </td>
<td>7581</td>
<td>&nbsp;</td>
<tr>
<td>Neutral Protease (Dispase)</td>
<td>Reagent</td>
<td>Worthington</td>
<td>LS02111</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

neuronal cell cultures from aplysia for high-resolution imaging of growth cones

Conos de crecimiento neuronales son estructuras altamente móviles en la punta de los axones que pueden detectar señales de orientación en el medio ambiente y la transducción de esta información en el movimiento direccional hacia la celda de destino adecuado. Para entender completamente cómo la información se transmite a la orientación de la superficie celular a la base de redes citoesqueleto dinámico, se necesita un sistema de modelo adecuado para imágenes de células vivas de la dinámica de proteínas de alta resolución temporal y espacial. Típico de los conos de crecimiento de vertebrados son demasiado pequeños para analizar cuantitativamente la F-actina y la dinámica de los microtúbulos. Las neuronas de la liebre de mar Aplysia californica son 5-10 veces más grande que las neuronas de los vertebrados, fácilmente se puede mantener a temperatura ambiente y son células muy robusta de micromanipulación y mediciones biofísicas. Sus conos de crecimiento tienen muy definido regiones citoplásmicas y un sistema de citoesqueleto bien descritos. Los cuerpos de las células neuronales se pueden microinyección con una variedad de sondas para el estudio de la motilidad del cono de crecimiento y orientación. En el presente Protocolo que demostrar un procedimiento para la disección de los ganglios abdominales, la cultura de las neuronas de células bolsa y la creación de una cámara de imágenes de imágenes de células vivas de los conos de crecimiento.

Watch this Scientific Journal Video about Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones at JoVE.com

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

Aplysia californica neuronas desarrollan un gran crecimiento en los conos de la cultura que son excelentes para imágenes de alta resolución de la motilidad del cono de crecimiento y orientación. Aquí, se presenta un protocolo para la disección y de las planchas de las neuronas de células Aplysia bolsa, así como para la creación de una cámara de imágenes de células vivas.

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 ...

neuronal-cell-cultures-from-aplysia-for-high-resolution-imaging

Research

JoVE Journal

Biology

11.3K vistas.  Purdue University. Aplysia californica neuronas desarrollan un gran crecimiento en los conos de la cultura que son excelentes para imágenes de alta resolución de la motilidad del cono de crecimiento y orientación. Aquí, se presenta un protocolo para la disección y de las planchas de las neuronas de células Aplysia bolsa, así como para la creación de una cámara de imágenes de células vivas.

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

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 ...

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. ...

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 ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Vídeo de alta resolución de Seguimiento de la locomoción en adultos de Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

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 ...

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 ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Generación de Dispersed presomitic mesodermo Cultivos Celulares for Imaging del pez cebra Segmentación del Reloj en células individuales

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

Método ex vivo de Imágenes de Alta Resolución de Motilidad Cilia en roedores las vías respiratorias epitelio

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Usando plusTipTracker Software a Medida microtúbulos dinámica en<em&gt; Xenopus laevis</em&gt; Conos de Crecimiento

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...