JB wurde von der Damon Runyon Cancer Research Foundation Fellowship DRG-1716 bis 1702 unterstützt. JZ wurde durch die NIH / NIGMS Fellowship F32 GM082174-02 unterstützt. NP ist ein Ermittler des Howard Hughes Medical Institute.

1. Vorbereitung fliegen Käfige <ol><li> Wachsen Drosophila (Wildtyp-Stämme wie Oregon-R oder Genotyp) in bequemen Behälter, wie 32-oz Insekten Tassen. </li><li> Transfer neu eclosed fliegt zu großen Embryo-Sammlung Käfigen oder fliegen Bevölkerung Käfigen. </li><li> Bewegen Sie den Käfigen in einen Raum mit einer Temperatur von ~ 25 ° C und Luftfeuchtigkeit bis 65% in einer leichten 12-h und einem dunklen 12-h-Zyklus, um die Sammlung von Synchron-Embryonen zu erleichtern. </li><li> Pflegen Sie die Fliegen in den Käfigen, indem man sie getötet Hefepaste auf Melasse-Platten ausgestrichen. Ändern Sie die Melasse-Platten (mit abgetöteten Hefe paste) einmal täglich für 2-3 d. </li></ol> 2. Sammlung von Synchron-Embryonen <ol><li> Am Tag der Primär-Zelle Vorbereitung, füttern die Fliegen wth frische Platten mit abgetöteten Hefe beschichtet für eine 1-2 h vor-lay Zeitraum einfügen. </li><li> Kurz vor dem 12-h Dunkel-Zyklus, ersetzen Sie die pre legen Platten mit frischen Melasse-Platten mit einer minimalen Menge von Hefe Paste ausgestrichen. Entsorgen Sie die pre lag Sammlung, die asynchrone älteren Embryonen enthält. </li><li> Sammeln Embryonen für einen Zeitraum von 1-2 h </li><li> Entfernen Sie die Platten mit den relativ synchron Bevölkerung von Embryonen und speichern bei 25 ° C für 5-6 h. Embryonen werden in den modernen Gastrulastadium zu diesem Zeitpunkt. </li></ol> 3. Sammlung und Reinigung von Embryonen <ol><li> Lösen Sie die Embryonen von den Platten mit einem nassen Pinsel, und anschließend gründlich mit lauwarmem Leitungswasser durch eine gestapelte Satz Siebe, so dass die Embryonen in die feinsten Sieb sammeln sich am Boden. Spülen Sie für ein paar Minuten, um so viel Hefe und fliegen Trümmer wie möglich zu entfernen. </li><li> Tipp der Siebe, so dass die Embryonen auf einer Seite sammeln und vorsichtig waschen Sie sie aus dem Sieb in eine dechorionation Korb. </li><li> Umzug in die Gewebekultur Haube Bereich, ohne dass die Embryonen austrocknen. </li></ol> 4. Homogenisierung von Embryonen und Beschichtung von Zellen <ol><li> Sterilisieren und dechorionate die Embryonen durch Eintauchen in 50% (vol / vol) Bleichmittel für 5-10 min. Spülen Sie den Embryonen von der Wand des Korbes mit 70% (vol / vol) Ethanol. Alternativ, waschen Sie die Bleiche aus Embryonen mit destilliertem Wasser, bevor Ethanol-Behandlung. Von diesem Zeitpunkt an Embryonen unter sterilen Bedingungen und in der Gewebekultur Haube behandelt werden. </li><li> Spülen Sie den Embryonen gründlich, um das Bleichmittel und / oder Ethanol mit autoklaviertem destilliertem Wasser zu entfernen. </li><li> Während der dechorionation Schritt füllen Dounce Homogenisator mit dem entsprechenden Volumen von Raumtemperatur M3 mittelfristig auf den Betrag von Embryonen basieren. Das Verhältnis zwischen dem Volumen des Embryos und der Medien sollten nicht mehr als 0,5-ml Embryonen werden: 40-ml-Medien (oder ~ 100-200 Embryonen / ml). </li><li> Legen Sie die gespülten Embryonen in einem sterilisierten Becher mit der Menge des M3 Medien genug, um tauchen Embryonen. Lassen Sie die Embryonen für 2-5 min einweichen. </li><li> Demontieren Sie die dechorionation Korb und blot die Nitex Mesh trocken sterilisiert Papierhandtücher. </li><li> Transfer der Embryonen in den Homogenisator mit einer sterilisierten Pinsel. </li><li> Homogenisieren vorsichtig mit kurzen Strichen mit einem losen Pistill ohne Erreichen der Boden des Röhrchens, bis alle Embryonen ausgesetzt sind. Dann vorsichtig aber fest, mit acht volle Takte zu homogenisieren. Nicht verdrehen Stößel, wie es oben und unten bewegt. </li><li> Übertragen Sie die Homogenat in eine sterile 50-ml konische Zentrifugenröhrchen entweder durch Gießen oder Pipettieren, und die Kappe der Röhre. </li><li> Zentrifuge für 10 min bei 40 g, Raumtemperatur Pellet die Gewebestücke, große Zellklumpen und vitelline Membranen. Übertragen Sie den Überstand mit den Zellen und Eigelb in ein sauberes Röhrchen und wiederholen Sie den Zentrifugationsschritt für 5 min. </li><li> Vorsichtig den Überstand durch Gießen oder Pipettieren in ein sauberes Röhrchen und drehen für 10 min bei 360g, Raumtemperatur die Zellen zu pelletieren. Überstand verwerfen. Zellpellet in 10 ml Kulturmedium (serum-freiem Medium für RNAi-Experimente). </li><li> Graf lebensfähigen Zellen mit einer Zählkammer die Zelldichte zu schätzen. In der Zwischenzeit, wiederholen Sie Schritt 4,10 bis Pellet den Zellen. </li><li> Resuspendieren der Zellen auf eine gewünschte Konzentration. Um zu beginnen, versuchen Sie einen Bereich von 1 ~ 5 x10 6 Zellen / ml und die Platte bei 1,7 bis 2,5 x10 5 Zellen / cm 2. </li></ol> 5. Primär-Zelle RNAi für Zellen, die auf eine 384-Well-Platte gewachsen <ol><li> Dispense 5 ul dsRNAs in jedes Well in einer Endkonzentration von ~ 250 ng pro Vertiefung einer 384-Well-Platte. </li><li> Verwenden Sie eine Mehrkanal-Pipette, verzichten 10 ml (1 ~ 4x106 Zellen / ml) von primären Zellen pro Vertiefung in einem serumfreien M3-Medium in einem 384-Well-Platte mit einem anderen dsRNA in jede Vertiefung. Alternativ können die Zellen auf 8-Well-Glas Kammer-Objektträgern für die konfokale Bildgebung kultiviert werden. </li><li> Centrifuge die Platten für 1 min bei 300g, Raumtemperatur. Kultur der Zellen in serum-freiem Medium bei 25 ° C für 22 h oder bei 18 ° C für 1-2 d. </li><li> 30 ml Serum-haltigem Kulturmedium in jede Vertiefung. </li&gt; <li> Centrifuge die Platten für 1 min bei 300g, Raumtemperatur. </li><li> Legen Sie die Platten in einer feuchten Kammer und Kultur die primären Zellen für weitere 5 ~ 7 d bei 25 ° C. </li><li> Bild der Zellen entweder live oder nach Immunfluoreszenzfärbung. </li></ol> 6. Repräsentative Ergebnisse: Bei optimalen Bedingungen werden die Zellen in der Kultur sehen mit einem runden Morphologie gesund. Die Kultur wird wenig Zelltrümmer, und 50 ~ 80% konfluente nach anfänglichen Beschichtung. Diese Zellen werden morphologische Veränderungen, nachdem sie über einen längeren Zeitraum hinweg kultiviert unterziehen. Normalerweise dauert es etwa 5-8 Tage für Zellen in der Kultur vollständig bei 25 ° C unterschieden werden Gute Qualität primären Kulturen sind in der Regel, wie gut die Zellen-of-Interest sind in der Kultur differenziert beurteilt. Zum Beispiel, primäre Muskelzellen haben oft eine Niederlassung Morphologie und werden vielkernigen Myotuben mit Streifen wie Myofibrillen, bestehend aus einer Reihe von Sarkomere. Voll differenzierte Myotuben wird in der Regel spontan in die Kultur zu bewegen. Im Gegensatz dazu bilden differenzierte primären Neuronen Cluster mit ihrem Zellkörper, und verbinden sich mit anderen Clustern mit ihrem Axon-Kabel. <img src="/files/ftp_upload/2215/2215fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Primäre Zellen durchlaufen morphologische Veränderungen, nachdem sie in der Kultur verchromt (A) A Hellfeldbild von primären Zellen nach rechts, nachdem er in eine Vertiefung einer 384-Well-Platte ausplattiert. Die Mehrzahl der Zellen sind rund und intakt und gut voneinander getrennt. (B) A Hellfeldbild der primären Zellkultur 20 Stunden nach dem Ausplattieren. Primäre Zellen bereits Cluster bilden (große Pfeilspitze) und die Muskeln mit einer Niederlassung Morphologie (langer Pfeil) kann in diesem Stadium erkannt werden. (C) Ein Phasenkontrast-Bild der primären Kultur 5 Tage nach dem Ausplattieren. Die Kultur sieht viel weiter fortgeschritten ist, mit neuronalen Erweiterungen (kleine Pfeile), neuronale Cluster (große Pfeile) und Muskeln (mittlere Pfeil). <img src="/files/ftp_upload/2215/2215fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Beispiele für primäre Zellen mit dsRNAs behandelt in einer 384-Well-Platte kultiviert Primäre Zellen wurden aus der Embryonen mit Dmef2-Gal4, D42-Gal4, UAS-mito-GFP-Transgene, in denen Dmef2-Gal4 und D42-Gal4-Laufwerk Ausdruck mito-GFP in den Muskeln und motorischen Neuronen bzw. isoliert. Die Zellen wurden mit dsRNAs gezielt entweder lacZ (AC) oder aufgeblasen (wenn) (DF) behandelt. Sowohl primäre Muskeln und Neuronen kann durch GFP (A, C, D, F) zu sehen. Die weißen Pfeile zeigen auf die neuronale Erweiterungen. Phalloidin-Färbung von Aktin (Pfeilspitzen) zeigt eine astähnlichen Muskulatur in der Kultur mit lacZ dsRNAs (B und C) behandelt, sondern Round-up Muskel-Morphologie in die Kultur mit, wenn behandelt dsRNAs (E und F). In der fusionierten Bilder (C, F), sind die Muskeln gelb gefärbt, aber rot negativen zeigt Neuronen und deren Erweiterungen (weiße Pfeile). <img src="/files/ftp_upload/2215/2215fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Konfokale Aufnahmen von primären Muskeln auf die menschliche Vitronektin-beschichteten Deckglas gleitet kultivierten Primäre Muskeln waren immunofluorescently mit anti-Titin Drosophila (KZ) (rot in der merge) und mit Phalloidin für Aktin (grün in der Zusammenführung) gefärbt. Die Top-Panels sind die Wildtyp-Kontrolle Muskel-und Unterseite diejenigen sind die schläge (CG31374) RNAi Muskel mit verkürzten Sarkomere.

<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>

Die Entwicklungsmuster der primären Kulturen von Drosophila-Zellen können sehr unterschiedlich sein, was möglicherweise auf die verschiedenen Variablen während der primären Zelle Herstellungsverfahren. Zunächst fliegt zur Eiablage verwendet werden soll jungen (innerhalb einer Woche alt) und gesund (frei von viralen oder bakteriellen Infektion). Unbefruchtete oder infizierten Embryonen müssen vermieden werden, da sie nutzlos und werden die Zellen aus den Embryonen nicht unterscheiden kann und wird nur fü...

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		<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>
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<td>Dounce tissue grinder,Kontes 40 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>KT885300-0100</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>
<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

Hier beschreiben wir ein Verfahren zur Herstellung und Kultivierung primärer Zellen losgelöst von Drosophila Gastrula Embryonen. In kurzen, inszeniert eine große Menge von Embryonen von jungen und gesunden Fliegen gesammelt, sterilisiert und dann physisch trennen in einer einzigen Zellsuspension mit einem Glas-Homogenisator. Nachdem er auf Kulturplatten oder Kammer-Objektträgern in angemessener Dichte an Kultur-Medium, können diese Zellen weiter in mehrere morphologisch-verschiedene Zelltypen, die durch ihre spezifischen Zell-Marker identifiziert werden können differenzieren. Darüber hinaus präsentieren wir die Bedingungen für die Behandlung dieser Zellen mit doppelsträngiger (ds) RNA Gen-Knockdown zu entlocken. Effiziente RNAi in Drosophila primären Zellen erfolgt durch einfaches Baden die Zellen in dsRNA-haltigen Kulturmedium erreicht. Die Fähigkeit zur Durchführung von effektiven RNAi Störung, zusammen mit anderen molekularbiologischen, biochemischen, Cell Imaging-Analysen erlauben eine Vielzahl von Fragen, die in Drosophila primären Zellen, insbesondere in Bezug auf differenzierte Muskel-und Nervenzellen beantwortet werden.

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

Primary Cell Cultures from Drosophila Gastrula Embryos

Wir bieten Ihnen ein detailliertes Protokoll zur Herstellung von primären Zellen losgelöst von<em&gt; Drosophil</em&gt; A Embryonen. Die Fähigkeit zur Durchführung der effektive RNAi Störung, zusammen mit anderen molekularbiologischen, biochemischen und bildgebenden Verfahren Zelle ermöglicht eine Vielzahl von Fragen, die in adressiert werden<em&gt; Drosophila</em&gt; Primärzellen.

Primären Zellkulturen aus Drosophila Gastrula Embryonen

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 Aufrufe.  Harvard Medical School. Wir bieten Ihnen ein detailliertes Protokoll zur Herstellung von primären Zellen losgelöst von Drosophil A Embryonen. Die Fähigkeit zur Durchführung der effektive RNAi Störung, zusammen mit anderen molekularbiologischen, biochemischen und bildgebenden Verfahren Zelle ermöglicht eine Vielzahl von Fragen, die in adressiert werden Drosophila Primärzellen.

Serie: 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.

Primäre neuronale Kulturen aus den Gehirnen von Late Stage Drosophila Puppen

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

Forschung

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.

Vorbereitung der neuronalen Kulturen aus Midgastrula Bühne Drosophila Embryonen

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.

Primäre Dissoziierte Mittelhirn Dopamin Zellkulturen aus Nager Neugeborene

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.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

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.

Tissue Gezielte Embryonale Chimären: Zebrafisch 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 ...

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.

Gründung Primary Adult Fibroblastenkulturen von Nagetieren

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.

Upright Imaging von Drosophila Embryos

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.

Imaging Zellgröße Veränderung der Lebens- Drosophila Embryos

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.

Isolierung und Reinigung von Kinesin aus Drosophila Embryonen

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.

Naive Adult Stem Cells Isolation von primären humanen Fibroblasten Kulturen

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