NZ gustaría agradecer a los miembros del laboratorio de Subhas Biswas, Andrew Sproul, y Willet Ryan por su formación en la disección y la cosecha de las neuronas simpáticas. Apoyado por becas de los NIH, NINDS.

1. Preparación para la disección: <ol><li> Seleccione la placa de cultivo apropiado (10 cm o placas de 6 pocillos o bien 24, etc) dependiendo de la naturaleza de sus experimentos, y el escudo con el colágeno de cola de rata-24 horas antes de la disección. Los métodos para la preparación de cola de rata colágeno y usarlo para placas de cultivo abrigo se han descrito en otra parte (1). </li><li> Prepare una solución de tripsina 0,25% en tampón fosfato salino (PBS) (sin EDTA) y el filtro de esterilización. Preparar alícuotas de 1 ml y almacenar a -20 º C. </li><li> Prepare una solución que contiene 2.5 mM de cada uno de uridina y 5 FDU-en agua destilada / desionizada H 2 O. Filtro a esterilizar y preparar alícuotas de 50 microlitros. Conservar a -20 º C. </li><li> Preparar medio completo: RPMI 1640 medio de cultivo con 10% inactivado por calor suero de caballo (el calor inactiva por incubación en un baño de agua a 56 º C durante 30 minutos) y el 5% de suero fetal bovino. </li><li> Prepare final Medium: RPMI 1640 medio de cultivo con 1% inactivado por calor suero de caballo + NGF (concentración 100ηg/ml final) + penicilina / estreptomicina (1 / 250) + 1 / 250 uridine/5-FDU solución (10 mM concentración final) . Nota: este medio debe estar compuesto por nuevos antes de su uso en todo momento. Para un experimento en un formato de placa de 24 pozos, uno tendrá que preparar 12 ml de este medio. Se recomienda que se prepare un poco más. NGF se pueden comprar en una variedad de fuentes comerciales (véase el cuadro al final de este protocolo). </li><li> Equipamiento: Un necesita un microscopio de disección y fuente de luz. Un iluminador de cuello de cisne de fibra óptica doble con puntas de enfoque es preferible. Además, será necesario limpiar y esterilizar (autoclave o por inmersión en etanol al 70% por lo menos 20 minutos) las herramientas de disección: dos pares de micro-disección pinzas de acero inoxidable, y un par de tijeras de disección (también de acero inoxidable y tanto desde Roboz). Dos agujas de jeringas estériles (26 gague x 1 ½ pulgadas o similar) también será necesario. Es necesario establecer una tabla de disección, que es un 3 &quot;x3&quot; pedazo de espuma de poliestireno envueltos en papel de aluminio y cubierta con una Kimwipe. Rocíe el tablero con etanol al 70% para descontaminar. Por último, la preparación del fuego pulido pipeta de vidrio de los pastos. Con el fin de pulir al fuego, tomar la pipeta de vidrio y sostenga la punta de la llama durante unos segundos, durante la rotación. Tras la inspección, la punta se verá más suave y la apertura será más estrecho. Esto es necesario para el paso en el que las células se disocian. Realizar fuego pulido en una campana de cultivos celulares para que la pipeta se mantiene estéril. </li></ol> 2. La disección del ganglio cervical superior: <ol><li> Recoger cachorros recién nacidos de ratas que están en edad de P0 o P1. </li><li> Limpie rápidamente el cachorro para la disección con el 70% de etanol para reducir las posibilidades de contaminación. </li><li> Rápidamente decapitar a las crías con un par de tijeras afiladas (este paso no se muestra en el video). Mantenga la cabeza con una pinza estéril contra una gasa estéril para drenar el exceso de sangre brevemente. </li><li> Coloque la cabeza sobre la almohadilla de la disección con la parte ventral hacia arriba y el extremo caudal orientada hacia usted. La tráquea cortada debe ser fácilmente visible. Use las agujas de jeringas estériles para asegurar la cabeza cortada a la plataforma de la disección de la siguiente manera: empujar una de las patas cuando el techo de la boca en la almohadilla y empuje el pasador de segundo, aunque la tráquea cortada en la almohadilla. Coloque la almohadilla bajo el microscopio de disección y comenzar la disección. </li><li> Usando ambas pinzas (una en cada mano) la piel clara de distancia y la grasa alrededor de la tráquea, teniendo cuidado de no profundizar demasiado. Haciendo esto expondrá un par de músculos que van casi en paralelo el uno al otro a ambos lados de la tráquea. Nota: durante la disección, tendrá que regar con agua fría o caliente PBS estéril, libre de suero RPMI 1640 que entrega con una pipeta Pasteur para quitar el exceso de sangre y para mantener el área limpia y húmeda. Esto se puede hacer sólo una vez o varias veces dependiendo de la velocidad se trabaja. Por un disector de la experiencia, sólo se tarda de dos a tres minutos para diseccionar un par de ganglios. </li><li> Use las pinzas para cortar y extirpar el músculo en un lado primero. </li><li> Una vez que el músculo se elimina, la arteria carótida se hará visible. A menudo, es la luz rosada y contiene la sangre. Puede aparecer un color más claro que otros vasos sanguíneos que puede ver en los alrededores y por esta razón, la arteria carótida puede no ser evidente de inmediato. Esta arteria corre a lo largo de la tráquea y se bifurca en lo que parece una &quot;Y&quot; en forma de cinta. Esta bifurcación es un hito importante y una vez que se encuentra, será relativamente fácil encontrar el SCG. </li><li> Cortar la arteria en el extremo más caudal y subirlo un poco con la pinza (el otro extremo todavía está conectado). Una vez hecho esto, podrás ver una forma de almendra, brillante en masa buscando pequeñas de tejido que se unen libremente a la arteria justo en el punto de bifurcación y que ha tread-como pre-y / o post-ganglionares de enlace. Este es el ganglio cervical superior. Use un segundo par de pinzas en la otra mano para separar suavemente y colóquelo en un plato de cultivo pequeño (35 mm) con frío, el suero libre de RPMI 1640. </li><li> A continuación, extraer el ganglio en el otro lado de la tráquea, siguiendo el mismo protocolo. </li></ol> 3. Limpieza de los ganglios: <ol><li> Una vez que todos los ganglios han sido diseccionados, que debería ser liberado lo más posible de tejido extraño. </li><li> Bajo el microscopio de disección, se puede ver que los ganglios se han unido las piezas de la arteria carótida, el tejido adiposo o de otro tipo. Trabajar con fórceps en cada mano para burlarse de distancia estos materiales no deseados. Lugar limpiar los ganglios en una estéril cónicos 15 ml, tubo de polipropileno, que contiene medio RPMI 1640. </li><li> Los conectores pre-y / o post-ganglionares también debe ser recortada. Cada ganglio tiene alrededor de 10.000 neuronas. Una vez que las neuronas están recubiertos, que volverá a crecer sus neuritas. </li></ol> 4. El establecimiento de la cultura neuronas simpáticas: <ol><li> Centrífuga de los ganglios en 200xg durante 2 minutos y descartar el sobrenadante. </li><li> Resuspender los ganglios en 0,5 a 1 ml de solución de tripsina 0,25%. Colocar en un baño de agua a 37 ˚ C o una incubadora durante 30 minutos. Esto le ayudará a disociar las células de los ganglios. </li><li> Después de 30 minutos, agregar 10 ml de medio completo para diluir y neutralizar la tripsina y centrifugar a 200xg durante 2 minutos. </li><li> Desechar el sobrenadante y resuspender en 2 ml de medio final. </li><li> Además se disocian las células de trituración de los ganglios arriba y hacia abajo con el fuego-pulido pipeta de vidrio de los pastos. Triturar los ganglios de 20 veces y colocar el tubo en hielo durante unos minutos. </li><li> Mientras tanto, el fuego a pulir la pipeta más para hacer la abertura más estrecha (aproximadamente 2 / 3 y 1 / 2 mm de diámetro). Espere unos minutos hasta que se enfríe y triturar los ganglios 20 veces más. A medida que continúe el medio serán cada vez más nublado que indica que las células se están disociados. Repita este paso una o dos veces más, dependiendo de lo bien que las células se disocian. Después de algún tiempo, te darás cuenta de que todos los ganglios se han disociado en las neuronas de los ganglios y no intacto son visibles en el tubo. Puede haber un poco de material tejido que no se disocian. En este punto, el tubo se sientan en el hielo durante unos minutos para que todos los residuos no disociado puede asentarse en el fondo. Ahora, recoger cuidadosamente casi todo el medio de la parte superior y colocar en otro tubo. Para asegurarse de que no recogen los restos sin disociar, deje unos 50 l en la parte inferior del tubo. Añadir más de medio final al tubo que contiene las células disociadas: este volumen depende del tipo de una placa de cultivo se utiliza para la cultura de las neuronas. Por ejemplo: si un plato de 24 y se va a emplear, a continuación, la placa de 0,5 ganglios por pozo (~ 10.000 neuronas por pocillo) en 0,5 ml de medio final por pocillo. Una camada de ratas típica consta de 12 crías, lo que daría 24 ganglios. Esto es suficiente para sembrar un 24 y placa y el volumen total de medio final es de 12 ml. Nota: los ganglios también contienen una pequeña población de células gliales y otros tipos de células que estarán presentes en la cultura. Utilizando Uridine/5-FDU en el medio final evitará su proliferación y promover su muerte. </li><li> Alimentación de las celdas cada 48 horas mediante la sustitución de 2 / 3 de la media con un fresco RPMI% suero de caballo + NGF y uridine/5-FDU. Nota: Al principio, la cultura contiene las neuronas simpáticas que mirar a su alrededor sin neuritas. Neuritas cortas se hacen visibles 24 horas después de la disección y se tornan progresivamente más denso y ramificado en el tiempo. </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. , (1998).</li><li>Biswas, S. C., Shi, Y., Sproul, A., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-apoptotic+Bim+Induction+in+Response+to+Nerve+Growth+Factor+Deprivation+Requires+Simultaneous+Activation+of+Three+Different+Death+Signaling+Pathways.">Pro-apoptotic Bim Induction in Response to Nerve Growth Factor Deprivation Requires Simultaneous Activation of Three Different Death Signaling Pathways.</a> The Journal of Biological Chemistry. 282 (40), 29368-29374 (2007).</li><li>Bocchini, V., Angeletti, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nerve+Growth+Factor:+purification+as+a+30,000-molecular-weight+protein.">The Nerve Growth Factor: purification as a 30,000-molecular-weight protein.</a> Proc. Natl. Acad. Sci. USA. (64), 787-794 (1969).</li><li>Dechant, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chat+in+the+Trophic+Web:+NGF+Activates+Ret+by+Inter-RTK+Signaling.">Chat in the Trophic Web: NGF Activates Ret by Inter-RTK Signaling.</a> Neuron. 33 (2), 156-158 (2002).</li><li>Deckwerth, T., Elliott, J. L., Knudson, C. M., Johnson, E. M., Snider, W. D., Korsmeyer, 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=Bax+is+required+for+neuronal+death+after+trophic+factor+deprivation+and+during+development.">Bax is required for neuronal death after trophic factor deprivation and during development.</a> Neuron. (17), 401-411 (1996).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+Vitro+Studies+on+the+Nerve+Growth+Factor+(NGF)+Requirement+of+Neurons+-+I.+Sympathetic+Neurons.">Quantitative in Vitro Studies on the Nerve Growth Factor (NGF) Requirement of Neurons - I. Sympathetic Neurons.</a> Developmental Biology. (58), 96-105 (1977).</li><li>Park, D. S., Morris, E. J., Padmanabhan, J., Shelanski, M. L., Geller, H. M., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin-dependent+kinases+participate+in+death+of+neurons+evoked+by+DNA-damaging+agents.">Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents.</a> The Journal of Cell Biology. 143, 457-467 (1998).</li><li>Pierchala, B. A., Ahrens, R. C., Paden, A. J., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+Growth+Factor+Promotes+the+Survival+of+Sympathetic+Neurons+through+the+Cooperative+Function+of+the+Protein+Kinase+C+and+Phosphatidylinositol+3-Kinase+Pathways.">Nerve Growth Factor Promotes the Survival of Sympathetic Neurons through the Cooperative Function of the Protein Kinase C and Phosphatidylinositol 3-Kinase Pathways.</a> The Journal of Cell Biology. 279, 27986-27993 (2004).</li></ol>

The authors have nothing to disclose.

Utilizamos ratas Sprague Dawley, de nuestras culturas. Tómese su tiempo y atención a la limpieza de los ganglios. Eliminar todos los restos, los vasos sanguíneos y grasa. Este paso es importante para asegurar una cultura que es más o menos homogénea y libre de los tipos de células extrañas. La adición de uridine/5-FDU gran medida eliminar cualquier resto de las células no neuronales (mitosis) contaminación de la cultura o al menos inhibir su proliferación. Además, durante el paso de la disociación, ser paciente ...

<!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>Forceps, Stainless steel #5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS4976</td>
<td>&nbsp;</td>
<tr>
<td>Scissors, DEAVER straight sharp-blunt- 43mm blades - 5.5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS6760</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 w/ L-Glutamin</td>
<td>Reagent</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>10-040-CV</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>SAFC Biosciences</td>
<td>12103c</td>
<td>&nbsp;</td>
<tr>
<td>Donor Horse Serum</td>
<td>Reagent</td>
<td>JRH Biosciences</td>
<td>12449</td>
<td>Heat-inactivate by incubating in 56&deg;C waterbath for 30 minutes.</td>
</tr>
<tr>
<td>Penicillin/streptomycin</td>
<td>Reagent</td>
<td>Gibco, Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin without EDTA</td>
<td>Reagent</td>
<td>Difco</td>
<td>MT 25-050-CI</td>
<td>&nbsp;</td>
<tr>
<td>Uridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>U3003</td>
<td>&nbsp;</td>
<tr>
<td>5-Fluoro-5' deoxyuridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F-8791</td>
<td>&nbsp;</td>
<tr>
<td>NGF</td>
<td>Reagent</td>
<td>Harlan Bioproduct</td>
<td>BT-5025</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Microscope and light source</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 ml polypropylene tubes</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes</td>
<td>Tool</td>
<td>Corning, Nunclone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

protocol for culturing sympathetic neurons from rat superior cervical ganglia (scg)

Los ganglios cervical superior (SCG) en las ratas son pequeños, brillantes, con forma de almendra estructuras que contienen las neuronas simpáticas. Estas neuronas proporcionan inervación simpática de la cabeza y el cuello y las regiones que constituyen una población bien caracterizados y relativamente homogénea (4). Las neuronas simpáticas dependen de factor de crecimiento nervioso (NGF) para el crecimiento de la supervivencia, diferenciación y axonal y de la amplia disponibilidad de NGF facilita su cultura y su manipulación experimental (2, 3, 6). Por estas razones, cultivos de neuronas simpáticas se han utilizado en una amplia variedad de estudios, incluyendo el desarrollo neuronal y la diferenciación, los mecanismos de muerte celular programada y patológica, y la transducción de señales (1, 2, 5 y 6). La disección de los SCG de ratas recién nacidas y las neuronas simpáticas de cultivo no es muy complicada y se puede dominar con bastante rapidez. En este artículo vamos a describir en detalle cómo diseccionar el SCG de crías de rata recién nacida y de utilizarlos para establecer cultivos de neuronas simpáticas. El artículo también se describen los pasos preparatorios y de los diversos reactivos y equipos que se necesitan para lograr este objetivo.

Watch this Scientific Journal Video about Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG at JoVE.com

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

Este es un protocolo que describe cómo aislar y cultivar primaria neuronas simpáticas de los ganglios cervical superior (SCG) de las crías recién nacido.

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

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

protocol-for-culturing-sympathetic-neurons-from-rat-superior-cervical

Research

JoVE Journal

Biology

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

22.7K vistas.  Columbia University. Este es un protocolo que describe cómo aislar y cultivar primaria neuronas simpáticas de los ganglios cervical superior (SCG) de las crías recién nacido.

Video: Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

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

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

Investigación

Biología

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Aislamiento de neutrófilos Protocolo

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Preparación y mantenimiento de las neuronas dorsales ganglios de raíz en las culturas con compartimientos

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

Introducción al Laboratorio de Worm: El cultivo de gusanos de Mutagénesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Un protocolo simple para la extracción de hemocitos de orugas silvestres

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

El cultivo primarios de rata medular interna recolección de células del conducto

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Toma de muestras de sangre de la vena lateral de la cola de la rata

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Aislamiento de CD146<sup&gt; +</sup&gt; Las células estromales mesenquimales de pulmón Residente de los pulmones de ratas

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Un protocolo simple de alta eficiencia para el aislamiento de islotes pancreáticos de ratones

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

Colección de biopsias de músculo esquelético del Compartimiento Superior del Musculus Humano Tibialis Anterior para la Evaluación Mecánica

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...