JB fue apoyada por el Damon Runyon Cancer Research Fellowship Foundation DRG-1716-02. JZ fue apoyado por el NIH / NIGMS F32 Fellowship GM082174-02. NP es un investigador del Instituto Médico Howard Hughes.

1. Preparación de las jaulas de vuelo <ol><li> Crecer Drosophila (cepas de tipo salvaje como Oregon-R o cualquier genotipo) en envases convenientes tales como tazas de insectos 32-oz. </li><li> Transferencia de las moscas recién eclosed a las grandes jaulas de recogida de embriones o volar jaulas población. </li><li> Mover las jaulas en una habitación con una temperatura de ~ 25 ° C y la humedad ~ 65% en una luz de 12 horas y un oscuro ciclo de 12 horas para facilitar la recolección de los embriones sincrónica. </li><li> Mantener a las moscas en las jaulas de la alimentación de ellos mató a pasta de levadura sembradas en placas de melaza. Cambiar las placas de melaza (con pasta de levadura muerta) una vez al día durante 2-3 d. </li></ol> 2. Recolección de los embriones síncrono <ol><li> El día de la primaria de preparación de células, se alimentan las moscas placas wth frescas bañadas en pasta de levadura muerto por un 1-2 h antes de poner punto. </li><li> Justo antes del ciclo de 12 horas de oscuridad, sustituir las placas de pre sentar con placas de melaza fresco rayado con una mínima cantidad de pasta de levadura. Deseche la colección pre sentar, que contiene asíncrono embriones más viejos. </li><li> Recoger los embriones durante un período de 1-2 h. </li><li> Quitar las placas que contienen a la población relativamente sincrónica de los embriones y guárdela a 25 ° C durante 5-6 h. Los embriones serán en la etapa avanzada de gástrula en este momento. </li></ol> 3. Recogida y lavado de embriones <ol><li> Afloje los embriones de las placas con un pincel húmedo, y enjuagar bien con agua corriente tibia a través de una serie de tamices apilados, de modo que los embriones se acumulan en el tamiz de malla más fina en la parte inferior. Enjuagar durante unos minutos para eliminar la levadura y los restos mucho volar como sea posible. </li><li> Consejo de los tamices para que los embriones se acumulan en un lado, y suavemente lave el tamiz en una cesta dechorionation. </li><li> Vaya al área de tejido campana cultura sin dejar que los embriones se sequen. </li></ol> 4. Homogeneización de los embriones y las células de revestimiento <ol><li> Esterilizar y dechorionate los embriones mediante la inmersión en un 50% (vol / vol) de cloro durante 5-10 minutos. Enjuague los embriones de la pared de la canasta con un 70% (vol / vol) de etanol. Por otra parte, lava con lejía de embriones con agua destilada antes del tratamiento de etanol. Desde este punto en adelante los embriones deben ser tratados en condiciones de esterilidad y en el capó de cultivo de tejidos. </li><li> Enjuague los embriones a fondo para eliminar el cloro y / o etanol con agua destilada en autoclave. </li><li> Durante el paso de dechorionation, llenar un homogeneizador Dounce con el volumen apropiado de medio M3 de la temperatura ambiente sobre la base de la cantidad de embriones. La relación entre el volumen de los embriones y la de los medios de comunicación no debe ser mayor de embriones de 0,5 ml: 40 ml de medios (o ~ 100-200 embriones / ml). </li><li> Colocar los embriones enjuagarse con un vaso de precipitados esterilizado con la cantidad de M3 medios de comunicación suficiente para sumergir los embriones. Deje que los embriones en remojo durante 2-5 min. </li><li> Desmontar la canasta dechorionation y seque la malla Nitex seca con toallas de papel esterilizado. </li><li> La transferencia de los embriones en el homogeneizador con un pincel esterilizados. </li><li> Homogeneizar suavemente con movimientos cortos y utilizando un mortero suelto, sin llegar a la parte inferior del tubo hasta que todos los embriones se suspenden. Luego, suavemente pero con firmeza, homogeneizar con ocho golpes por completo. No tuerza la mano del mortero, ya que se mueve hacia arriba y hacia abajo. </li><li> Transferir el homogeneizado en una solución estéril de 50 ml tubo de centrífuga cónico, ya sea por vertido o pipeta, y la tapa del tubo. </li><li> Centrifugar durante 10 minutos a 40 g, la temperatura ambiente para que sedimenten los restos de tejido, grandes grupos de células y las membranas vitelina. Transferir el sobrenadante que contiene las células y la yema de un tubo limpio y repetir el paso de centrifugación durante 5 min. </li><li> Transferir cuidadosamente el sobrenadante mediante el vertido o pipeta en un tubo limpio y girar durante 10 minutos a 360 gramos, la temperatura ambiente para precipitar las células. Eliminar el sobrenadante. Resuspender el botón celular en 10 ml de medio de cultivo (medio libre de suero para los experimentos de RNAi). </li><li> Recuento de células viables mediante un hemocitómetro para estimar la densidad de las células. Mientras tanto, repita el paso 4.10 para precipitar las células. </li><li> Resuspender las células a la concentración deseada. Para empezar, pruebe con un rango de 1 ~ 5 x 10 6 células / ml y la placa a cabo en 1,7 a 2,5 x10 5 células / cm 2. </li></ol> 5. Primaria de las células RNAi para las células cultivadas en una placa de 384 pocillos <ol><li> Prescindir de 5 l de dsRNAs en cada pocillo a una concentración final de ~ 250 ng por pocillo en una placa de 384 pocillos. </li><li> Utilice una pipeta multicanal, dispensar 10 ml (1 ~ 4x106 células / ml) de pilas por pocillo en un medio libre de suero M3 en una placa de 384 pocillos que contiene un dsRNA diferentes en cada pozo. Alternativamente, las células pueden ser cultivadas en 8 y portaobjetos de vidrio de cámara para imagen confocal. </li><li> Centrifugar las placas durante 1 minuto a 300 g, la temperatura ambiente. Cultivar las células en medio libre de suero a 25 ° C durante 22 horas o menos 18 ° C durante 1-2 d. </li><li> Añadir 30 ml de medio de cultivo que contiene suero a cada pocillo. </li&gt; <li> Centrifugar las placas durante 1 minuto a 300 g, la temperatura ambiente. </li><li> Coloque las placas en una cámara húmeda y la cultura de las células primarias durante 5 ~ 7 días a 25 ° C. </li><li> Imagen de las células ya sea en directo o después de la tinción de inmunofluorescencia. </li></ol> 6. Los resultados representativos: En condiciones óptimas, las células de la cultura un aspecto saludable, con una morfología de todo el año. La cultura tendrá restos de células pequeñas, y de 50 a 80% confluente después de la siembra inicial. Estas células sufren cambios morfológicos después de haber sido cultivadas durante un período prolongado de tiempo. Por lo general, tarda unos 5-8 días para que las células en la cultura para ser totalmente diferenciadas a 25 ° C. Culturas de buena calidad primaria suelen ser juzgados por la forma en las células de interés se diferencian en la cultura. Por ejemplo, las células musculares primarias a menudo tienen una morfología similar a la rama y se convierten en miotubos multinucleados con banda-como las miofibrillas que consiste en una serie de sarcómeros. Miotubos completamente diferenciadas por lo general se mueven de forma espontánea en la cultura. Por el contrario, diferenciadas neuronas primarias formar grupos con sus cuerpos celulares, y conectarse con otros grupos con los cables de su axón. <img src="/files/ftp_upload/2215/2215fig1.jpg" alt="Figura 1" /> Figura 1. Las células primarias sufren cambios morfológicos después de ser plateado en la cultura (A) Una imagen de campo claro de pilas después de que se colocaron en un pocillo de una placa de 384 pocillos. La mayoría de las células son redondas y intacta y bien separadas. (B) Una imagen brillante campo de cultivo de células primarias 20 horas después de la siembra. Las células primarias ya formar grupos (grandes punta de flecha) y los músculos con una morfología similar a la rama (flecha larga) se pueden reconocer en esta etapa. (C) Una imagen de contraste de fases de cultivo primario 5 días después de la siembra. La cultura se ve mucho más avanzado, con extensiones neuronal (flechas pequeñas), los grupos neuronales (puntas de flecha grande) y músculos (de tamaño mediano flecha). <img src="/files/ftp_upload/2215/2215fig2.jpg" alt="Figura 2" /> Figura 2. Ejemplos de células primarias tratados con dsRNAs cultivaron en una placa de 384 pocillos Las células primarias fueron aisladas de los embriones que Dmef2-Gal4, D42-Gal4, UAS-mito-GFP transgenes, en el que Dmef2-Gal4 y D42-Gal4 expresión de la unidad de mito-GFP en los músculos y las neuronas motoras, respectivamente. Las células fueron tratadas con dsRNAs objetivo sea lacZ (AC) o exageradas (si) (DF). Tanto los músculos principales y las neuronas pueden ser vistos por las buenas prácticas agrarias (A, C, D, F). Las flechas blancas señalan las extensiones de las neuronas. Tinción phalloidin de actina (puntas de flecha) revela una estructura músculo-sucursal como en el cultivo tratado con lacZ dsRNAs (B y C), pero vuelta a la morfología del músculo en el cultivo tratado con si dsRNAs (E y F). En las imágenes fusionadas (C, F), los músculos son manchas amarillas, pero el rojo neuronas muestra negativa y sus extensiones (flechas blancas). <img src="/files/ftp_upload/2215/2215fig3.jpg" alt="Figura 3" /> Figura 3. Micrografías confocal de los músculos principales cultivos en la salud humana vitronectina revestida de diapositivas cubierta de vidrio Principales músculos fueron teñidas con anti-immunofluorescently Drosophila Titin (KZ) (rojo en la mezcla) y con phalloidin para actina (verde en la fusión). Los paneles superiores son el control muscular de tipo salvaje y las de abajo son los préstamos para ajuste estructural (CG31374) músculo RNAi con sarcómeros acortados.

<ol>
	<li>Ashburner, M., Roote, J., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory culture of Drosophila. , 588-599 (1999).</li><li>Bai, J., Hartwig, J. H., Perrimon, N. S. A. L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+WH2-domain-containing+protein,+promotes+sarcomeric+actin+filament+elongation+from+pointed+ends+during+Drosophila+muscle+growth.">a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth.</a> Dev. Cell. 13, 828-842 (2007).</li><li>Bai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+screening+in+Drosophila+primary+cells+for+genes+involved+in+muscle+assembly+and+maintenance+.">RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance .</a> Development. 135, 1439-1449 (2008).</li><li>Sepp, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+outgrowth+genes+using+genome-wide+RNAi.">Identification of neural outgrowth genes using genome-wide RNAi.</a> PloS. Genet. 4, e1000111-e1000111 (2008).</li><li>Donady, J. J., Fyrberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+culturing+of+Drosophila+embryonic+cells+in+vitro.">Mass culturing of Drosophila embryonic cells in vitro.</a> TCA Manual. 3, 685-687 (1977).</li><li>Seecof, R. L., Alleaume, N., Teplitz, R. L., Gerson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+neurons+and+myocytes+in+cell+cultures+made+from+Drosophila+gastrulae.">Differentiation of neurons and myocytes in cell cultures made from Drosophila gastrulae.</a> Exp. Cell Res. 69, 161-173 (1971).</li><li>Bai, J., Sepp, K. J., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Drosophila+primary+cells+dissociated+from+gastrula+embryos+and+their+use+in+RNAi+screening.">Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening.</a> Nature Protocols. 4, 1502-1512 (2009).</li></ol>

El patrón de desarrollo de los cultivos primarios de células de Drosophila puede variar mucho, posiblemente debido a las diversas variables durante el proceso de preparación de células primarias. En primer lugar, las moscas utilizadas para la puesta de huevos deben ser pequeños (menos de una semana de edad) y sanos (libres de la infección viral o bacteriana). Embriones fertilizados o infectado se debe evitar, ya que son inútiles, y las células derivadas de los embriones que no se puede diferenciar y só...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Shields and Sang M3 medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3652</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>JRH Biosciences</td>
<td>12103-500M</td>
<td>&nbsp;</td>
<tr>
<td>Insulin from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-6634</td>
<td>&nbsp;</td>
<tr>
<td>Large embryo collection cages</td>
<td>&nbsp;</td>
<td>Genesee Scientific</td>
<td>59-101</td>
<td>&nbsp;</td>
<tr>
<td>#25 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-454</td>
<td>&nbsp;</td>
<tr>
<td>#45 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-462</td>
<td>&nbsp;</td>
<tr>
<td>#120 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-474</td>
<td>&nbsp;</td>
<tr>
<td>Dounce tissue grinder, Wheaton 7 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>62400-620</td>
<td>&nbsp;</td>
<tr>
<td>Dounce tissue grinder,Kontes 40 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>KT885300-0040</td>
<td>&nbsp;</td>
<tr>
<td>Dounce tissue grinder,Kontes 40 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>KT885300-0100</td>
<td>&nbsp;</td>
<tr>
<td>Costar, 384-well plate</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>29444-078</td>
<td>&nbsp;</td>
<tr>
<td>Lab-Tek II Chambered coverglass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>171080</td>
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primary cell cultures from drosophila gastrula embryos

Aquí se describe un método para la preparación y el cultivo de células primarias disociada de Drosophila embriones gástrula. En resumen, una gran cantidad de embriones de moscas de la escena joven y saludable son recolectados, esterilizados, y luego físicamente disocia en una suspensión de células individuales usando un homogeneizador de vidrio. Después de estar chapada en placas de cultivo o la cámara de diapositivas en una densidad adecuada en medio de cultivo, estas células pueden diferenciarse en más de varios tipos de células morfológicamente distintas, que pueden ser identificados por sus marcadores celulares específicos. Además, se presentan las condiciones para el tratamiento de estas células con doble cadena (ds) RNA para obtener caída de genes. Eficiente ARNi en células Drosophila primaria se logra simplemente impregnando las células en medio de cultivo que contienen dsRNA. La capacidad para llevar a cabo eficaz perturbación RNAi, junto con otros molecular, análisis bioquímicos, imágenes de células, que permiten una variedad de preguntas a ser respondidas en las células de Drosophila primaria, especialmente las relacionadas con musculares diferenciadas y las células neuronales.

Watch this Scientific Journal Video about Primary Cell Cultures from Drosophila Gastrula Embryos at JoVE.com

Primary Cell Cultures from Drosophila Gastrula Embryos

Ofrecemos un protocolo detallado para la preparación de las células primarias disociada de<em&gt; Drosophil</em&gt; A los embriones. La capacidad de llevar a cabo la efectiva perturbación RNAi, junto con otros métodos de imagen molecular, bioquímica y celular permite una variedad de cuestiones que deben abordarse en<em&gt; Drosophila</em&gt; Células primarias.

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 ...

primary-cell-cultures-from-drosophila-gastrula-embryos

Research

JoVE Journal

Biology

Primary Cell Cultures from Drosophila Gastrula Embryos

13.8K vistas.  Harvard Medical School. Ofrecemos un protocolo detallado para la preparación de las células primarias disociada de Drosophil A los embriones. La capacidad de llevar a cabo la efectiva perturbación RNAi, junto con otros métodos de imagen molecular, bioquímica y celular permite una variedad de cuestiones que deben abordarse en Drosophila Células primarias.

Video: Primary Cell Cultures from Drosophila Gastrula Embryos

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 ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

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

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

Primaria disociada Mesencéfalo culturas dopamina celulares de roedores recién nacidos

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

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

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

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

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

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

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Tejido diana quimeras embrionarias: pez cebra Gástrula Trasplante de células

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Establishing Primary Adult Fibroblast Cultures From Rodents

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

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

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

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

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imagen de la posición vertical Drosophila Los embriones

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Celular imagen Cambiar forma de vida Drosophila Los embriones

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Aislamiento y purificación de Kinesin desde Drosophila Los embriones

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

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

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

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

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

Naïve Adult Stem Cells Aislamiento de Primaria cultivos de fibroblastos humanos

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