JB a été soutenue par la Damon Runyon Cancer Research Fellowship Foundation DRG-1716-1702. JZ a été soutenu par le NIH / NIGMS Bourse F32 GM082174-02. NP est un enquêteur de l&#39;Institut médical Howard Hughes.

1. Préparation des cages volée <ol><li> Cultivez la drosophile (souches sauvages tels que l&#39;Oregon-R ou tout autre génotype) dans des contenants pratiques tels que des tasses d&#39;insectes 32-oz </li><li> Transfert mouches nouvellement eclosed à de grandes cages d&#39;embryon de collecte ou de voler des cages de la population. </li><li> Déplacer les cages dans une pièce avec une température de ~ 25 ° C et une humidité ~ 65% dans une lumière de 12 h et une noire de 12 h du cycle afin de faciliter la collecte d&#39;embryons synchrone. </li><li> Maintenir les mouches dans les cages en les nourrissant tué pâte de levure striés sur des plaques de la mélasse. Changer les plaques de la mélasse (la levure à la pâte de tués) une fois par jour pendant 2-3 d. </li></ol> 2. Collecte des embryons synchrones <ol><li> Le jour de l&#39;élément primaire de préparation, nourrir les mouches wth revêtement des plaques fraîches avec de la levure a tué la pâte pour un 1-2 h de pré-lay période. </li><li> Juste avant le cycle de 12 h d&#39;obscurité, de remplacer les plaques pré couché avec plaques mélasse douce strié avec une quantité minimale de pâte levure. Jeter la collection pré laïcs, qui contient asynchrones embryons âgés. </li><li> Recueillir des embryons pour une période de 1-2 h. </li><li> Retirez les plaques contenant de la population relativement synchrones d&#39;embryons et les conserver à 25 ° C pendant 5-6 h. Les embryons seront au stade avancé gastrula à ce moment. </li></ol> 3. Collecte et lavage des embryons <ol><li> Desserrer les embryons à partir des plaques avec un pinceau humide, et rincez abondamment à l&#39;eau du robinet tiède à travers une série de tamis empilés, de sorte que les embryons de recueillir dans le tamis plus fins treillis au fond. Rincer pendant quelques minutes pour enlever autant que la levure et les débris volent que possible. </li><li> Astuce du tamis afin que les embryons s&#39;accumulent d&#39;un côté, et les laver en douceur du tamis dans un panier dechorionation. </li><li> Déplacer vers la zone de culture de tissu capuche sans laisser les embryons se dessécher. </li></ol> 4. Homogénéisation des embryons et des cellules de plaquage <ol><li> Stériliser et dechorionate les embryons en les immergeant dans 50% (vol / vol) javel pendant 5-10 min. Rincez les embryons sur le mur du panier avec 70% (vol / vol) d&#39;éthanol. Sinon, laver l&#39;eau de Javel hors embryons avec l&#39;eau distillée avant traitement à l&#39;éthanol. De ce point embryons partir doivent être manipulés dans des conditions stériles et dans la hotte de culture de tissus. </li><li> Rincez les embryons à fond pour enlever l&#39;eau de Javel et / ou de l&#39;éthanol avec de l&#39;eau distillée en autoclave. </li><li> Pendant l&#39;étape dechorionation, remplissez un homogénéisateur Dounce avec le volume approprié de milieu M3 température ambiante en fonction du montant des embryons. Le rapport entre le volume des embryons et celle des médias ne devraient pas être trop embryons de 0,5 ml: 40 ml, les médias (ou ~ 100 à 200 embryons / ml). </li><li> Placer les embryons rincés dans un bécher stérilisés avec le montant de M3 assez de journalistes pour submerger les embryons. Laissez tremper pendant les embryons 2-5 min. </li><li> Démonter le panier dechorionation et éponger le filet Nitex sécher avec des serviettes en papier stérilisé. </li><li> Transfert des embryons dans l&#39;homogénéisateur avec un pinceau stérilisé. </li><li> Homogénéiser doucement avec des mouvements courts en utilisant un pilon lâches sans atteindre le fond du tube jusqu&#39;à ce que tous les embryons sont suspendus. Puis doucement, mais fermement, homogénéiser avec huit coups plein. Ne pas tourner le pilon comme il se déplace de haut en bas. </li><li> Transférer le broyat dans un stérile de 50 ml, tube à centrifuger conique soit par versement ou de pipetage, et le bouchon du tube. </li><li> Centrifuger pendant 10 min à 40g, la température ambiante pour obtenir un culot de débris de tissu, des bouquets à grandes cellules et les membranes vitelline. Transférer le surnageant contenant les cellules et le jaune dans un tube propre et répétez l&#39;étape de centrifugation pendant 5 min. </li><li> Soigneusement transférer le surnageant par pipetage versant ou dans un tube propre et de spin pendant 10 min à 360g, la température ambiante à un culot cellulaire. Jeter le surnageant. Resuspendre la cellule dans 10 ml de milieu de culture granulés (milieu sans sérum pour des expériences de RNAi). </li><li> Nombre de cellules viables en utilisant un hémocytomètre pour estimer la densité cellulaire. Pendant ce temps, répétez l&#39;étape 4.10 pour culotter les cellules. </li><li> Resuspendre les cellules à une concentration souhaitée. Pour commencer, essayez une fourchette de 1 ~ 5 x10 6 cellules / ml et une plaque à moins 1.7 au 2.5 x10 5 cellules / cm 2. </li></ol> 5. Primaire à cellules ARNi pour les cellules cultivées sur une plaque de 384 puits <ol><li> Distribuer 5 pl de dsRNA dans chaque puits à une concentration finale de ~ 250 ng par puits dans une plaque de 384 puits. </li><li> Utiliser une pipette multi-canaux, distribuer 10 ml (1 ~ 4x106 cellules / ml) de cellules primaires par puits dans un milieu sans sérum M3 dans une plaque de 384 puits contenant un ARNdb différents dans chaque puits. Alternativement, les cellules peuvent être cultivées sur 8-même diapositives chambre de verre pour l&#39;imagerie confocale. </li><li> Centrifuger les plaques pendant 1 min à 300g, la température ambiante. Culture des cellules dans un milieu sans sérum à 25 ° C pendant 22 h ou à 18 ° C pendant 1-2 d. </li><li> Ajouter 30 ml de milieu de culture contenant du sérum à chaque puits. </li&gt; <li> Centrifuger les plaques pendant 1 min à 300g, la température ambiante. </li><li> Placer les plaques dans une chambre humide et la culture des cellules primaires pour un supplément de 5 ~ 7 jours à 25 ° C. </li><li> Image des cellules soit en direct ou après coloration par immunofluorescence. </li></ol> 6. Les résultats représentatifs: Dans des conditions optimales, les cellules dans la culture sera une apparence saine avec une morphologie ronde. La culture aura peu de débris cellulaires, et de 50 ~ 80% confluentes après étalement initial. Ces cellules vont subir des changements morphologiques, après avoir été cultivés pendant une période de temps prolongée. Cela prend habituellement environ 5-8 jours pour les cellules de la culture pour être pleinement différenciées à 25 ° C. Bonne qualité des cultures primaires sont généralement jugés par la façon dont les cellules d&#39;intérêt sont différenciées en culture. Par exemple, les cellules musculaires primaires ont souvent une morphologie semblable à la branche et de devenir des myotubes multinucléés avec une bande-like myofibrilles composé d&#39;une série de sarcomères. Myotubes différenciés seront entièrement habituellement spontanément passer dans la culture. En revanche, les neurones primaires différenciées forment des grappes avec leur corps cellulaire, et se connecter avec d&#39;autres clusters avec leurs câbles axone. <img src="/files/ftp_upload/2215/2215fig1.jpg" alt="Figure 1" /> Figure 1. Les cellules primaires subissent des changements morphologiques, après avoir été plaqué dans la culture (A) Une image en champ clair de cellules primaires à droite après avoir été plaqué dans un puits d&#39;une plaque de 384 puits. La majorité des cellules sont rondes et intacte et bien séparés. (B) Une image en champ clair de la culture de cellules primaires 20 heures après placage. Les cellules primaires déjà former des amas (grosse flèche) et les muscles avec une morphologie semblable à la branche (flèche longue) peuvent être reconnus à ce stade. (C) Une image en contraste de phase de la culture primaire 5 jours après l&#39;étalement. La culture ressemble beaucoup plus avancé, avec des extensions neuronales (petites flèches), des grappes de neurones (pointes de flèches grand) et les muscles (moyennes flèche). <img src="/files/ftp_upload/2215/2215fig2.jpg" alt="Figure 2" /> Figure 2. Des exemples de cellules primaires traités avec dsRNA culture dans une plaque de 384 puits Les cellules primaires ont été isolés à partir des embryons porteurs Dmef2-Gal4, D42-Gal4, UAS-GFP mito-transgènes, dans lequel Dmef2-Gal4 et D42-Gal4 l&#39;expression de lecteur de mito-GFP dans les muscles et les neurones moteurs, respectivement. Les cellules ont été traitées avec dsRNA ciblant soit lacZ (AC) ou gonflés (si) (DF). Les deux principaux muscles et les neurones peut être vu par la GFP (A, C, D, F). Les flèches blanches point à l&#39;prolongements neuronaux. Phalloïdine coloration de l&#39;actine (flèches) révèle une structure musculaire branche comme dans la culture traitée avec lacZ dsRNA (B et C), mais la morphologie musculaire round-up dans la culture traitée avec si dsRNA (E et F). Dans les images fusionnées (C, F), les muscles sont colorés en jaune, mais rouge négative neurones montre et leurs extensions (flèches blanches). <img src="/files/ftp_upload/2215/2215fig3.jpg" alt="Figure 3" /> Figure 3. Micrographies confocale des principaux muscles humains cultivés sur vitronectine lames recouvertes couvercle en verre Muscles primaires ont été colorés avec immunofluorescently anti-drosophile Titin (KZ) (rouge dans la fusion) et avec la phalloïdine pour l&#39;actine (vert à la fusion). Les panneaux supérieurs sont les muscles de contrôle de type sauvage et ceux du bas sont la Sals (CG31374) musculaire ARNi avec des sarcomères raccourcie.

<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>

Le schéma de développement des cultures primaires de cellules de drosophile peut varier considérablement, probablement due à des variables de plusieurs cours du processus de préparation de cellules primaires. Tout d&#39;abord, les mouches utilisées pour la ponte doivent être jeunes (dans une semaine) et saine (exempte d&#39;infection virale ou bactérienne). Embryons non fécondés ou infectés doivent être évités, car ils sont inutiles et des cellules dérivées de ces embryons ne peuvent pas se diff...

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<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>
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<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>
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<td>#45 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-462</td>
<td>&nbsp;</td>
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<td>#120 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-474</td>
<td>&nbsp;</td>
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<td>Dounce tissue grinder, Wheaton 7 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>62400-620</td>
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<td>Dounce tissue grinder,Kontes 40 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>KT885300-0040</td>
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<td>Dounce tissue grinder,Kontes 40 mL</td>
<td>&nbsp;</td>
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<td>Costar, 384-well plate</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>29444-078</td>
<td>&nbsp;</td>
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<td>Lab-Tek II Chambered coverglass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
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primary cell cultures from drosophila gastrula embryos

Nous décrivons ici une méthode de préparation et de la culture de cellules primaires dissociées à partir d&#39;embryons de drosophile gastrula. En bref, une grande quantité d&#39;embryons mis en scène des mouches jeunes et sains sont collectées, stérilisée, puis physiquement dissocié en une suspension cellulaire unique à l&#39;aide d&#39;un homogénéiseur de verre. Après avoir été plaqué sur des plaques de culture ou des diapositives de chambre à une densité appropriée dans le milieu de culture, ces cellules peuvent se différencier en d&#39;autres de plusieurs types de cellules morphologiquement distinctes et qui peuvent être identifiés par leurs marqueurs cellulaires spécifiques. Par ailleurs, nous présentons les conditions pour le traitement de ces cellules avec double brin (ds) ARN pour susciter inactivation génique. Efficace ARNi dans les cellules de drosophile primaire est accomplie par la simple baignant les cellules dans un milieu de culture contenant ARNdb. La capacité de mener efficacement une perturbation ARNi, avec d&#39;autres moléculaire, analyses biochimiques, d&#39;imagerie cellulaire, permettra une variété de questions à répondre dans les cellules de drosophile primaire, en particulier ceux liés à la musculaires différenciées et des cellules neuronales.

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

Primary Cell Cultures from Drosophila Gastrula Embryos

Nous fournissons un protocole détaillé pour la préparation de cellules primaires dissociées de<em&gt; Drosophil</em&gt; Un embryon. La capacité à mener à bien la perturbation effective ARNi, avec d&#39;autres méthodes d&#39;imagerie moléculaire, biochimique et cellulaire permettra une variété de questions qui seront abordées dans les<em&gt; Drosophile</em&gt; Cellules primaires.

Cultures de cellules primaires à partir Drosophile Embryons Gastrula

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

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JoVE Journal

Biology

Primary Cell Cultures from Drosophila Gastrula Embryos

13.8K vues.  Harvard Medical School. Nous fournissons un protocole détaillé pour la préparation de cellules primaires dissociées de Drosophil Un embryon. La capacité à mener à bien la perturbation effective ARNi, avec d'autres méthodes d'imagerie moléculaire, biochimique et cellulaire permettra une variété de questions qui seront abordées dans les Drosophile Cellules primaires.

Vidéo: 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.

Cultures neuronales primaires du cerveau des drosophiles stade tardif pupes

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

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Biologie

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.

Préparation des cultures de neurones d&#39;embryons de drosophile Midgastrula scène

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.

Primaire Dissocié cultures Mésencéphale dopamine cellulaire du nouveau-né contre les rongeurs

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 préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

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.

Tissu ciblé chimères embryonnaires: Zebrafish cellulaire Gastrula 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 ...

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.

Établir des cultures primaires de fibroblastes adultes à partir de rongeurs

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.

Imagerie verticale de<em&gt; Drosophila</em&gt; Embryons

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.

Changement de forme d&#39;imagerie cellulaire en vie Drosophile Embryons

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.

Isolement et purification de la kinésine de Drosophila Embryons

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 cellules souches adultes Isolation de cultures de fibroblastes humains primaires

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

Methods Article

Cultures de cellules primaires à partir Drosophile Embryons Gastrula