Agradecemos a los miembros del laboratorio de Gelfand que contribuyeron al desarrollo de estos protocolos. Las investigaciones realizadas en esta publicación fue apoyada por el Instituto Nacional de Ciencias Médicas Generales de los Institutos Nacionales de la Salud bajo el número de concesión R01GM052111 a VIG y por el Departamento del Gobierno Vasco de Educación, Universidades e Investigación con el número premio BFI-2011-295 a UdC.

1. Células S2 de Drosophila <ol><li> Preparación de cubreobjetos recubiertos de ConA <ol><li> Coloque cubreobjetos en un bastidor de cerámica y lavarlos por inmersión en ácido crómico durante 1 hora. [ATENCION: El ácido crómico es corrosivo y puede provocar irritación de los ojos, la nariz, la garganta y la piel]. </li><li> Enjuagar a fondo con cubreobjetos en constante funcionamiento dH 2 O durante 30 minutos hasta que el ácido está completamente lavada. </li><li> Deje que entre aire cubreobjetos secos y recubrirlos con ConA (solución de 0,5 mg / ml en dH 2 O) durante 30 min. </li><li> Enjuague cubreobjetos con dH 2 O durante 15 minutos y dejar secar al aire. Cubreobjetos ConA recubiertos pueden ser almacenados hasta por 1 mes. </li></ol></li><li> Plating las células <ol><li> Permitir que las células S2 a crecer de forma exponencial en 2 matraces T25 o T75 cm dependiendo del número de células requeridas para el experimento. </li><li> Cuente la densidad de células utilizando un hemocitómetro. Añadir 1 ml de crecimiento medio (por ejemplo Insectos-Xpress) en un 35 mm de placa de cultivo de tejidos con cubreobjetos recubiertos con ConA, y suavemente transferir ~ 1 x 10 5 células a la placa. Esta densidad celular es óptima para la imagen porque los procesos de diferentes células no se superponen. </li><li> Con el fin de inducir la formación de procesos, inmediatamente después de la siembra añadir 1 ml de medio con 5 mM CytoD al plato (a la concentración final de 2,5 M CytoD). Permitir el desarrollo completo de los procesos mediante la incubación de las células por lo menos durante 2 horas a 25 º C antes de formación de imágenes. </li></ol></li></ol> 2. Drosophila Primaria Neurona Cultura <ol><li> Preparación para la cultura de las neuronas <ol><li> Preparar Drosophila medio de crecimiento neuronal (medio de Schneider suplementado con: suero 20% bovino fetal, inactivado por calor a 55 º C durante 30 min; 5 mg / ml de insulina; 100 g / ml de penicilina, 100 mg / ml de estreptomicina; 10 mg / ml de tetraciclina) . Este apoyomedio de complementado Schneider se puede almacenar a 4 º C durante 6 meses. </li><li> Preparar manzana viales de agar jugo de recogida de embriones de Drosophila (22,5 g de agar, 12,0 g de sacarosa y 750 ml de H 2 O en un matraz de 2 litros; autoclave durante 20-25 minutos, añadir 250 ml de zumo de manzana y verter ~ 10 ml en cada Drosophila vial, después de la solidificación, enchufe viales con bolas de algodón, y envolver los viales con un envoltorio de plástico para alimentos para evitar el exceso de secado de los alimentos). El jugo de manzana en frascos de agar se puede almacenar a 4 º C en bolsas de plástico bien cerrados durante 4 semanas. </li><li> Coat 25 mm cubreobjetos circulares ácido-lavado con ConA (ver detalles en el Protocolo para las células Drosophila S2) y esterilizar bajo luz ultravioleta durante 10 a 15 min. Estos cubreobjetos se pueden almacenar durante un mes. </li></ol></li><li> La cultura de las neuronas embrionarias de Drosophila <ol><li> Mantenga joven adulto vuela en manzana viales de agar jugo suplementados con levadura seca durante 1-2 días antes de la recolección de los embriones fo la preparación de la neurona. </li><li> En el día de la recolección, con el fin de sincronizar los embriones recogidos, la transferencia de moscas adultas a una manzana fresca jugo vial agar durante 1 hora precolección a la luz y desechar estos embriones. </li><li> Transferencia vuela a un nuevo vial de agar jugo de manzana, recoger los embriones durante 1 hora, y la transferencia de las moscas a otro vial durante otra hora, tanto en la oscuridad para aumentar el rendimiento de embriones. </li><li> Retire las moscas adultas en circulación vial agar jugo de manzana y dejar que los embriones se desarrollan otras 4 horas para las etapas 9-11 a temperatura ambiente (4-6 embriones-hr de edad son entre la etapa 9-11). </li><li> Retire los embriones por chorros de agua en viales y aflojando los embriones del agar jugo de manzana con un pincel pequeño (al menos 10 embriones son necesarios para una buena preparación de la neurona). </li><li> La transferencia de los embriones a un 100 m de malla de nylon filtro de células con una pipeta de transferencia para drenar el agua (prefregado la pipeta de transferencia en Drosophila tampón de lavado de embriones, el 0,4% de NaCl, 0,03% TritónX-100). </li><li> Recoger los embriones de la coladera usando un pequeño pincel, y transferirlos en un microtubo de 1.5 ml llena con 1 ml de lavado de embriones de Drosophila (Nota: limpiar las fibras de agar o de algodón con el pincel o la punta de la pipeta). En este protocolo, el 1,5 ml microtubos viene con un juego maja pellet desechable. </li><li> Lave los embriones en Drosophila lavado de embriones de 3 aumentos. </li><li> Dechorionate los embriones en una solución fresca 1:01 de lejía comercial y etanol al 95% durante 10 min [PRECAUCIÓN: lejía puede causar irritación en los ojos, la nariz, la garganta y la piel]. </li><li> Mover a la campana de cultivo de tejidos, y lavar los embriones 2x en medio de Schneider complementado. </li><li> Deja 200-300 l medio en el tubo con los embriones, y suavemente se disocian mecánicamente embriones dechorionated usando Kimble Chase majas pellets desechables que han sido esterilizadas con etanol al 70% (moler 10-15x). </li><li> Centrifugar la mezcla se homogeneizó a 20 xg durante 2 min, y TRAnsfer el sobrenadante a un tubo nuevo. [Este paso es opcional, sino que ayuda a eliminar los desechos del embrión] </li><li> Centrifugar el sobrenadante a 550 xg durante 2 min para sedimentar las células. </li><li> Resuspender el sedimento en 500 l complementado medianas y centrifugado células de Schneider de nuevo a 550 xg durante 2 min, repetir el ciclo de la resuspensión de hilatura de una vez. </li><li> Finalmente resuspender el precipitado en ~ 100 l complementado medio de Schneider y placa las células sobre un portaobjetos recubierto con ConA 25 mm en un 35 mm placa de Petri estéril. Incubar durante 10 min para permitir la unión celular. </li><li> Añadir 2 ml de medio suplementado de Schneider, con 5 mM CytoD para sumergir el cubreobjetos con células unidas. </li><li> Incubar el plato en un humidificado incubadora de 25 º C durante la noche antes de exponer. </li></ol></li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Aquí presentamos los protocolos detallados para estudiar el transporte de carga dependiente de microtúbulos en las células S2 de Drosophila y la cultura de la neurona primaria. Ambos procesos y neuritas S2 son estructuras llenas de paquetes de microtúbulos arrays que sirven como pistas para el transporte de orgánulos. Dos estrategias se utilizan típicamente a orgánulos de la etiqueta: la transfección de vectores que codifican una proteína fluorescente con una secuencia de o...

Células S2 de Drosophila han ganado cada vez más popularidad para el estudio de muchos procesos celulares. Estas células se obtuvieron originalmente de tripsinizadas embriones en fase tardía de OregonR Drosophila melanogaster y mantienen características similares a los macrófagos 1. Células S2 de Drosophila ofrecen muchas ventajas sobre otras líneas celulares. Ellos se cultivan a 25 ° C sin CO 2, y se pueden obtener imágenes para muchas horas a temperatura ambiente sin la necesidad de calefacción o el intercambio de gases. Más importante aún, las células S2 de Drosophila son muy sensibles al tratamiento de RNAi desmontables permitiendo alta eficiencia de proteínas de interés. Células S2 son altamente transfectables y líneas celulares estables se pueden seleccionar en menos de cuatro semanas para permitir la expresión ectópica estable de proteínas de interés. S2 células crecen normalmente bien vagamente unidos pero no se diseminan en un frasco o en suspensión. Ellos pueden ser inducidas a propagarse en cubreobjetos recubiertos previamente con un Conca lectina plantaNavalin A (ConA). La periferia de las células propagadas es especialmente fina y por lo tanto es ideal para microscopia de células vivas. Protocolos detallados para el cultivo las células S2, el tratamiento de RNAi, las imágenes en directo y de inmunotinción se pueden encontrar en la literatura 2,3. Nuestro laboratorio ha adoptado células S2 como un sistema modelo para estudiar el transporte de carga a base de microtúbulos. Células S2 de Drosophila tratados con cualquier fármaco que despolimeriza o corta la F-actina, tales como citocalasina D (CytoD), crecen los procesos largos llenos de microtúbulos uniformemente polarizadas 4 -10, lo que lo convierte en un sistema ideal para estudiar el movimiento de carga a lo largo de los microtúbulos arrays. Diferentes parámetros de transporte en estos procesos (es decir. Trayectorias longitudes, velocidades, direccionalidad, etc) se pueden medir fácilmente utilizando el software de partículas de seguimiento automatizado, DiaTrack (Semasopht: http://www.microscopy.info/Organization/Details/4423; Dr. . Pascal Vallotton: http://www.csiro.au/en/Organisation-Structure/Divisions/Mathematics-Informatics-and-Statistics/PascalVallotton.aspx # A4). Estas propiedades hacen que las células S2 un sistema atractivo para el estudio de transporte de carga a lo largo de los microtúbulos en células de cultivo de tejidos. Los experimentos piloto en células S2 se pueden extender a un modelo fisiológicamente importante de los estudios de transporte de carga en las neuronas primarias de Drosophila. De manera similar a las células S2, neuronas cultivadas a partir de embriones disociadas son permeables a las moléculas pequeñas tales como colorantes y los inhibidores de, y de alta resolución de imágenes en vivo de orgánulos de transporte se pueden realizar en las neuronas a temperatura ambiente. Usando Drosophila con diferentes antecedentes genéticos, podemos examinar la función de genes en las neuronas primarias por nocaut (mutaciones genéticas de pérdida de la función), caída (inyección de dsRNA o expresión) o la expresión ectópica (moscas transgénicas). En concreto, la fragmentación de los filamentos de actina utilizando tratamiento CytoD no impide el crecimiento de neuritas, sino que crecen más rápido, y por lo tanto podemos estudiar microtúbulos Dependeorgánulos de transporte NT en las neuronas tratadas con CytoD sin la influencia de los filamentos de actina 11. Por lo tanto, los cultivos primarios de neuronas combinan las ventajas de las células de cultivo de tejidos y vuelan genética, lo que hace que sea un gran sistema para estudiar el transporte de carga en un sistema fisiológico relevante 10,12. Dado que algunas enfermedades neurodegenerativas familiares podrían ser causados ​​por o asociados con defectos de transporte de carga (por ejemplo, la enfermedad de Parkinson 13, la enfermedad de Huntington 14, la enfermedad de Alzheimer 15,16, la esclerosis lateral amiotrófica / ALS 17, síndrome HMN7B y Perry 18,19 y el tipo de ataxia espinocerebelosa 5/SCA5 20), cultivos primarios de neuronas pueden ser útiles en el análisis de los efectos de las mutaciones específicas que causan estas enfermedades en el transporte dependiente de microtúbulos. Por último, las propiedades importantes de transporte dependiente de microtúbulos están bien conservadas entre los mamíferos y las moscas, pero <em&gt; Cultura genética y de células de Drosophila es menos costoso y consume mucho tiempo, lo que justifica el uso de células de Drosophila cultivadas para estudiar los aspectos esenciales de orgánulos de transporte en condiciones normales y patológicas.

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			<td height="61" style="height:61px;width:124px;">Powder Insect cell medium (Schneider&#8217;s)</td>
			<td style="width:133px;">Sigma&#160;</td>
			<td style="width:179px;">S9895</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Insect &#8211;Xpress medium</td>
			<td style="width:133px;">Fisher</td>
			<td style="width:179px;">BW12730Q</td>
		</tr>
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			<td height="21" style="height:21px;width:124px;">Insulin</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">I6634</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Penicillin</td>
			<td style="width:133px;">Research Products International</td>
			<td style="width:179px;">P93000</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Streptomycin</td>
			<td style="width:133px;">Research Products International</td>
			<td style="width:179px;">S62000</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Tetracycline hydrochloride</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">T7660-5G</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Fetal bovine serum</td>
			<td style="width:133px;">Atlanta Biologicals</td>
			<td style="width:179px;">S11150</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Concanavalin A</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">C2010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Cytochalasin D</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">IC15077105</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">LysoTracker Red DND-99</td>
			<td style="width:133px;">Molecular Probes</td>
			<td style="width:179px;">L7528</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">100 &#181;m Cell strainer</td>
			<td style="width:133px;">Fisher Scientific</td>
			<td style="width:179px;">22363549</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">25 mm Circle cover glass</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">48380 080</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Drosophila Vials</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">89092-730</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:124px;">Disposable pellet pestles with matching microtubes</td>
			<td style="width:133px;">Kimble Chase</td>
			<td style="width:179px;">749520-0000</td>
		</tr>
	</tbody>
</table>

organelle transport in cultured drosophila cells: s2 cell line and primary neurons.

Células S2 de Drosophila sembraron en un cubreobjetos en la presencia de cualquier largos procesos no ramificados forma de fármaco actina-despolimerización de microtúbulos llenas de manera uniforme polarizadas. Orgánulos se mueven a lo largo de estos procesos a través de los motores de microtúbulos. Fácil mantenimiento, alta sensibilidad a la proteína mediada por RNAi knock-down y un procedimiento eficaz para la creación de líneas celulares estables que hacen las células S2 de Drosophila un sistema modelo ideal para estudiar el transporte de carga por la imagen en vivo. Los resultados obtenidos con las células S2 se pueden aplicar además a un fisiológicamente más relevantes del sistema: el transporte axonal en las neuronas primarias cultivadas a partir de embriones de Drosophila disociadas. Neuronas cultivadas crecen neuritas largas llenas de microtúbulos agrupados, muy similares a los procesos de S2. Al igual que en las células S2, orgánulos en neuronas cultivadas pueden ser visualizados por cualquiera de los tintes fluorescentes-orgánulo específico o mediante el uso de marcadores de orgánulos fluorescentes codificadas por ADN inyectado en embriones tempranos o expresado en transgenic vuela. Por lo tanto, el transporte de orgánulos se puede grabar fácilmente en neuronas cultivadas en cubreobjetos de vidrio utilizando imágenes viviente. Aquí se describen los procedimientos para el cultivo y la visualización de transporte de carga en las células S2 de Drosophila y neuronas primarias. Creemos que estos protocolos hacen ambos sistemas accesibles para los laboratorios que estudian el transporte de carga.

Células Drosophila S2 adjunta en la propagación cubreobjetos recubiertos de ConA en un plano y redondo en forma de &quot;panqueque&quot; (Figuras 1A y 1C). Sin embargo, cuando las células S2 sembraron en cubreobjetos en la presencia de CytoD y permitir que se extienda durante 2-3 h, forman procesos largos y delgados ocupados por los microtúbulos paralelos (Figuras 1B y 1D). Estos microtúbulos tienen polaridad uniforme con plus-termina a <s...

Watch this Scientific Journal Video about Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons. at JoVE.com

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Células S2 de Drosophila y neuronas cultivadas son grandes sistemas de obtención de imágenes de orgánulos de transporte con motor in vivo. Aquí se describen los protocolos detallados para el cultivo de ambos tipos de células, su imagen y el análisis de los transportes.

Orgánulos Transporte en Cultivadas<em&gt; Drosophila</em&gt; Celdas: S2 Línea Celular y neuronas primarias.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

organelle-transport-cultured-drosophila-cells-s2-cell-line-primary

Research

JoVE Journal

Biology

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

13.6K vistas.  Feinberg School of Medicine, Northwestern University.  Células S2 de Drosophila y neuronas cultivadas son grandes sistemas de obtención de imágenes de orgánulos de transporte con motor in vivo. Aquí se describen los protocolos detallados para el cultivo de ambos tipos de células, su imagen y el análisis de los transportes. 

Video: Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

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

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Imágenes en vivo del denso núcleo de vesículas en la Primaria neuronas del hipocampo cultivadas

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

Preparación de Drosophila Células S2 para microscopía de luz

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Aislamiento y cultivo primario de células hepáticas de ratas

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagen Ca Local<sup&gt; 2 +</sup&gt; Las señales en las células de mamífero en cultivo

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a &quot;Humanized&quot; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Generación de un &quot;humanizado&quot;<em&gt; Drosophila</em&gt; S2 Línea Celular Sensible a farmacológico Inhibición de la Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Medición de la Absorción de Glucosa y Respuesta a la Estimulación de la Insulina en<em&gt; In Vitro</em&gt; Myotubes primario humano cultivado

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

Ensayo de co-inmunoprecipitación utilizando proteínas nucleares endógenas de las células cultivadas bajo condiciones hipóxicas

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

Electrofisiología de células enteras de células enterocromafines murino cultivadas primario

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...