Wir danken den Mitgliedern des Gelfand Labor, die zur Entwicklung dieser Protokolle beigetragen. Forschung in dieser Veröffentlichung berichtet, wurde durch das National Institute of General Medical Science der National Institutes of Health unter Verleihungsnummer R01GM052111 an die VIG und baskischen Regierung Ministerium für Bildung, Hochschulen und Forschung unter Verleihungsnummer BFI-2011-295 bis UDC unterstützt.

1. Drosophila S2-Zellen <ol><li> Herstellung von ConA beschichtete Deck <ol><li> Zeigen Deckgläser in einem Keramik-Zahnstange und waschen Sie sie durch Chromsäure Eintauchen für 1 Stunde. [ACHTUNG: Chromsäure ist ätzend und kann zu Reizungen von Augen, Nase, Rachen und Haut verursachen]. </li><li> Deckgläser gründlich mit ständig laufenden dH 2 O für 30 Minuten, bis die Säure vollständig ausgewaschen. </li><li> Lassen Deckgläser der Luft trocknen und überziehen sie mit ConA (0,5 mg / ml Lösung in dH 2 O) für 30 min. </li><li> Spülen Deckgläser mit dH 2 O für 15 Minuten und lassen Sie sie an der Luft trocknen. ConA-beschichtete Deckgläser bis zu 1 Monat gelagert werden. </li></ol></li><li> Ausplattieren der Zellen <ol><li> S2-Zellen zu ermöglichen, exponentiell in Abhängigkeit von der Anzahl von Zellen für das Experiment erforderlich wachsen T25 oder T75-cm 2-Kolben. </li><li> Zählen Sie die Zelldichte mit einer Zählkammer. 1 ml Wachstums medium (zB Insect-Xpress) in eine 35-mm-Gewebekulturschale mit ConA-beschichtete Deckglas, und sanft über ~ 1 x 10 5 Zellen in die Schale. Diese Zelldichte ist optimal für Abbildungsverfahren, weil aus verschiedenen Zellen nicht überlappen. </li><li> Um die Bildung von Prozessen zu veranlassen, unmittelbar nach dem Plattieren 1ml Medium mit 5 uM CytoD zu der Schale (bis zu einer Endkonzentration von 2,5 uM CytoD). Ermöglichen eine vollständige Entwicklung von Verfahren durch Inkubation der Zellen mindestens 2 h bei 25 º C vor der Bilderzeugung. </li></ol></li></ol> 2. Drosophila Primär Neuron Kultur <ol><li> Vorbereitung für Neuronenkultur <ol><li> Vorbereitung Drosophila neuronalen Wachstumsmedium (Schneider-Medium, ergänzt mit 20% fötalem Rinderserum, Wärme bei 55 º C für 30 min inaktiviert, 5 ug / ml Insulin, 100 ug / ml Penicillin, 100 ug / ml Streptomycin, 10 ug / ml Tetracyclin) . Diese supergänzt Schneider Medium kann bei 4 º C für 6 Monate gelagert werden. </li><li> Bereiten Apfelsaft-Agar Fläschchen für das Sammeln von Drosophila-Embryonen (22,5 g Agar-Agar, 12,0 g Saccharose und 750 ml H 2 O in einem 2-Liter-Kolben, Autoklaven für 20-25 Minuten, fügen Sie 250 ml Apfelsaft und gießen ~ 10 ml in jede Drosophila Fläschchen, nach der Erstarrung, stecken Fläschchen mit Watte, und wickeln Sie Flaschen mit Plastikfolie über dem Trocknen der Lebensmittel zu vermeiden). Der Apfelsaft in Agar Fläschchen können bei 4 º C in gut verschlossenen Plastikbeutel für 4 Wochen gelagert werden. </li><li> Coat säuregewaschen 25 mm Kreis Deckgläser mit ConA (siehe Details im Protokoll für die Drosophila-S2-Zellen) und sterilisieren sie unter UV-Licht für 10-15 min. Diese Deck kann für einen Monat gelagert werden. </li></ol></li><li> Drosophila embryonalen Neuronenkultur <ol><li> Halten junger Erwachsener fliegt in Apfelsaft-Agar-Röhrchen mit Trockenhefe ergänzt für 1-2 Tage vor dem Sammeln Embryonen foder Neuron Vorbereitung. </li><li> Am Tag sammeln, um die gesammelten Embryonen zu synchronisieren, fliegt Transfer Erwachsenen zu einem frischen Apfelsaft-Agar Fläschchen für 1 Stunde in Precollection Licht und entsorgen diese Embryonen. </li><li> Über fliegt zu einer neuen Flasche Apfelsaft-Agar, sammeln Embryonen für 1 Stunde, und übertragen die Fliegen zu einem anderen Fläschchen für eine weitere Stunde, sowohl in dunklen, um den Embryo Ausbeute zu erhöhen. </li><li> Entfernen Sie die erwachsenen Fliegen aus der Apfelsaft-Agar-Röhrchen und lasse die Embryonen entwickeln, weitere 4 h bis 11.09 Stufen bei Raumtemperatur (4-6 h alten Embryonen sind zwischen Bühne 9-11). </li><li> Entfernen Sie die Embryonen durch Spritzen von Wasser in Flaschen und Lösen der Embryonen aus der Apfelsaft-Agar mit einem kleinen Pinsel (mindestens 10 Embryonen werden für eine gute Vorbereitung Neuron erforderlich). </li><li> Übertragen Sie die Embryonen auf einen 100 um Nylon-Mesh Zellsieb mit einer Transferpipette Wasser abzulassen (Vorspülen die Transferpipette in Drosophila-Embryo Waschpuffer, 0,4% NaCl, 0,03% TritonX-100). </li><li> Sammeln Sie die Embryonen aus dem Sieb mit einem kleinen Pinsel, und übertragen Sie sie in ein 1,5 ml Mikroröhrchen mit 1 ml Wasch Drosophila-Embryo (Hinweis: Reinigen keine Agar-oder Baumwollfasern mit dem Pinsel oder Pipettenspitzen) gefüllt. In diesem Protokoll wird das 1,5 ml-Mikroröhrchen mit einem passenden Einweg Pellets zerstoßen. </li><li> In Drosophila-Embryo Wasch 3x waschen Embryonen. </li><li> Dechorionate die Embryonen in einer frischen 1:1-Lösung von Handelsbleichmittel und 95% Ethanol für 10 min [ACHTUNG: Bleichmittel kann zu Reizungen von Augen, Nase, Rachen und Haut verursachen]. </li><li> Gehen Sie zur Gewebekultur Kapuze, und waschen Embryonen in 2x ergänzt Schneider Medium. </li><li> Lassen 200-300 ul Medium in der Röhre mit den Embryonen, und sanft mechanisch distanzieren dechorionated Embryonen mit Kimble Chase Einweg-Pellet Stößel, die von 70% Ethanol sterilisiert worden sind (schleifen 10-15x). </li><li> Zentrifugieren der homogenisierten Mischung bei 20 × g für 2 min und transfer der Überstand in ein frisches Röhrchen. [Dieser Schritt ist optional, es hilft, um Schmutz zu entfernen Embryo] </li><li> Zentrifugieren des Überstandes bei 550 g für 2 min Zellen zu pelletieren. </li><li> Das Pellet in 500 ul wieder ergänzt Schneider mittel-und Spin-Zellen nach unten bei 550 g für 2 min; wiederholen Sie die Aufwirbelung Spinnzyklus einmal. </li><li> Schließlich das Pellet in 100 ul ~ ergänzt Schneider-Medium und der Platte der Zellen auf einer 25 mm ConA-beschichtete Deckglas in einem sterilen 35 mm Petrischale. Inkubation für 10 min auf die Zellhaftung zu ermöglichen. </li><li> 2 ml ergänzt Schneider-Medium, mit 5 &amp;mgr; M CytoD auf das Deckglas mit anhaftenden Zellen einzutauchen. </li><li> Inkubieren Sie die Schale in einem befeuchteten 25 ° C Inkubator über Nacht vor der Bildgebung. </li></ol></li></ol>

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Die Autoren erklären, dass sie keine finanziellen Interessen konkurrieren.

Hier präsentieren wir Ihnen ausführliche Protokolle für die Untersuchung der Mikrotubuli-abhängige Frachtverkehr in Drosophila S2-Zellen und primäre Neuronenkultur. Beide S2 Prozesse und Neuriten sind Strukturen von gebündelten Mikrotubuli-Arrays, die als Spuren für Organellentransport dienen gefüllt. Zwei Strategien werden in der Regel Label Organellen verwendet: Transfektion von Vektoren ein fluoreszierendes Protein kodiert, mit einem Organell-Targeting-Sequenz oder Färbu...

Drosophila S2-Zellen haben sich zunehmender Beliebtheit für das Studium vieler zellulärer Prozesse gewonnen. Diese Zellen wurden ursprünglich aus Trypsin späten Stadium Embryonen von Drosophila melanogaster OregonR erhalten und zu pflegen Makrophagen-ähnliche Funktionen ein. Drosophila S2-Zellen bieten viele Vorteile gegenüber anderen Zelllinien. Sie kultiviert bei 25 ° C ohne CO 2, und kann für viele Stunden bei Raumtemperatur abgebildet werden, ohne die Notwendigkeit einer Erwärmung oder Gasaustausch. Noch wichtiger ist, Drosophila S2-Zellen sind sehr empfindlich auf RNAi-Behandlung ermöglicht hohe Effizienz Knockdown von Proteinen von Interesse. S2-Zellen sind sehr transfizierbar und stabile Zelllinien können in weniger als vier Wochen gewählt werden, um stabile ektopische Expression von Proteinen von Interesse zu ermöglichen. S2-Zellen normal wachsen entweder lose befestigt, aber nicht auf einen Kolben oder in Suspension zu verbreiten. Sie können bewogen, auf Deckgläsern mit einer Pflanze Lektin conca schwemmt verteilt werdennavalin A (ConA). Der Umfang der ausgebreiteten Zellen ist besonders dünn und ist somit ideal für Lebendzellmikroskopie. Detaillierte Protokolle für die S2-Zellen Kultur, RNAi-Behandlung, Live-Licht-Bildgebung und Immunfärbung in der Literatur 2,3 gefunden werden. Unser Labor hat S2-Zellen als Modellsystem, um Mikrotubuli-basierte Frachtverkehr zu studieren. Drosophila S2-Zellen mit jedem Medikament, das depolymerisiert oder trennt F-Actin, wie Cytochalasin D (CytoD) behandelt, wachsen lange Prozesse einheitlich polarisiert Mikrotubuli 4 gefüllt angenommen -10, die es zu einem idealen System für Fracht Bewegung entlang Mikrotubuli studieren. Verschiedene Parameter des Verkehrs in diesen Prozessen (. Dh Trajektorien Längen, Geschwindigkeiten, Richtungs etc) leicht mit automatisierten Partikel-Tracking-Software, DiaTrack (Semasopht gemessen werden: http://www.microscopy.info/Organization/Details/4423, Dr. . Pascal Vallotton: http://www.csiro.au/en/Organisation-Structure/Divisions/Mathematics-Informatics-and-Statistics/PascalVallotton.aspx # a4). Diese Eigenschaften machen S2-Zellen eine ansprechende System für die Untersuchung Frachtverkehr entlang von Mikrotubuli in Gewebekulturzellen. Pilotversuche in S2-Zellen kann zu einer physiologisch wichtigen Modell der Frachttransportstudien in Drosophila primären Neuronen erweitert werden. Ähnlich S2-Zellen sind Neuronen aus dissoziierten Embryonen kultiviert durchlässig für kleine Moleküle wie Farbstoffe und Inhibitoren und hochauflösende Live-Darstellung von Organellen-Transport in Nervenzellen bei Raumtemperatur durchgeführt werden. Mit Drosophila mit unterschiedlichen genetischen Hintergründen können wir Gen-Funktion in primären Neuronen untersuchen, durch Knockout (genetische loss-of-function Mutationen), Zuschlag (dsRNA Injektion oder Ausdruck) oder ektopische Expression (transgene Linie). Insbesondere ist die Fragmentierung von Aktin-Filamenten mit CytoD Behandlung nicht verhindert Neuritenwachstum, sondern sie schneller wachsen und somit können wir Mikrotubuli-depende studierennt Organellentransport in CytoD behandelten Neuronen ohne den Einfluss von Aktin-Filamenten 11. Daher primären neuronalen Kulturen verbinden die Vorteile von Gewebekulturzellen und fliegen Genetik, das es ein großartiges System für Frachttransport in einem physiologisch relevanten System 10,12 studieren. Da mehrere familiäre neurodegenerative Erkrankungen durch verursacht werden oder mit Frachttransportdefekte (zB Parkinson-Krankheit 13 in Verbindung gebracht werden, Huntington-Krankheit 14, Alzheimer-Krankheit 15,16, Amyotrophe Lateralsklerose / ALS 17, HMN7B und Perry-Syndrom 18,19 und Spinocerebellar Ataxie Typ 5/SCA5 20) können primären neuronalen Kulturen nützlich bei der Analyse der Wirkungen von bestimmten Mutationen, die diese Krankheiten auf Mikrotubuli-abhängigen Transport sein. Schließlich werden wichtige Eigenschaften der Mikrotubuli-abhängigen Transport auch zwischen Säugetieren und Fliegen erhalten, aber <em&gt; Drosophila Gen-und Zellkultur ist weniger teuer und zeitaufwendig ist, rechtfertigt die Verwendung von kultivierten Drosophila-Zellen für die Untersuchung wesentlichen Aspekte der Organellentransport in normalen und pathologischen Bedingungen.

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

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

Drosophila-S2-Zellen auf einem Deckglas überzogen in das Vorhandensein von Aktin-Depolymerisation Arzneiform lange unverzweigte Prozesse einheitlich polarisiert Mikrotubuli gefüllt. Organellen bewegen sich entlang dieser Prozesse durch Mikrotubuli-Motoren. Einfache Wartung, hohe Empfindlichkeit für RNAi-vermittelten Protein-knock-down und effiziente Verfahren für die Erstellung von stabilen Zelllinien machen Drosophila S2-Zellen ein ideales Modellsystem für Frachttransport durch Live-Bildgebung zu untersuchen. Axonalen Transport in primären Neuronen von Drosophila-Embryonen kultiviert dissoziiert: Die mit S2-Zellen erhaltenen Ergebnisse weiter zu einer physiologisch relevanten System angewendet werden. Kultivierten Neuronen wachsen lange Neuriten mit gebündelten Mikrotubuli, sehr ähnlich S2 Prozesse gefüllt. Wie in S2-Zellen, Organellen kann in kultivierten Neuronen entweder durch Organell-spezifischen Fluoreszenzfarbstoffen oder durch Verwendung von fluoreszierenden Organellen-Marker von DNA kodiert visualisiert werden in frühen Embryonen injiziert oder in transgen exprimiertic fliegt. Daher kann Organelltransport leicht in Neuronen auf Deckgläser mit lebendigen Bild kultiviert aufgezeichnet werden. Hier beschreiben wir Verfahren zur Kultivierung und Visualisierung Frachtverkehr in Drosophila S2-Zellen und primäre Neuronen. Wir glauben, dass diese Protokolle beide Systeme zugänglich für Labore Studium Frachtverkehr zu machen.

Drosophila-S2-Zellen auf der ConA-beschichtete Deckglas Ausbreitung in eine flache runde &quot;Pfannkuchen&quot;-Form (1A und 1C) angebracht. Wenn jedoch S2-Zellen auf Deckgläsern ausplattiert in Gegenwart CytoD und für 2-3 Stunden verteilt, bilden sie lange dünne Prozesse parallel Mikrotubuli (Fig. 1B und 1D) gefüllt. Diese Mikrotubuli einheitliche Polarität mit Plus-Enden aus 7. Zeitraffer-Fluoreszenz-Imaging von Drosophil...

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

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

Drosophila S2-Zellen und kultivierten Neuronen sind große Systeme für die Bildgebung von motorbetriebenen Organellentransport in vivo. Hier beschreiben wir ausführliche Protokolle für die Kultivierung beider Zelltypen, die Abbildung und Analyse des Verkehrs.

Organell-Transport in kultivierten<em&gt; Drosophila</em&gt; Zellen: S2-Zelllinie und primären Neuronen.

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

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

Research

JoVE Journal

Biology

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

13.6K Aufrufe.  Feinberg School of Medicine, Northwestern University.  Drosophila S2-Zellen und kultivierten Neuronen sind große Systeme für die Bildgebung von motorbetriebenen Organellentransport in vivo. Hier beschreiben wir ausführliche Protokolle für die Kultivierung beider Zelltypen, die Abbildung und Analyse des Verkehrs. 

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

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

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

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

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

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

Live-Imaging von Dense-Core-Vesikel in Primary hippokampalen Neuronen

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

Preparation of Drosophila S2 cells for Light Microscopy

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

Preparation of Drosophila S2 cells for Light Microscopy

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

Vorbereitung der Drosophila S2-Zellen für Lichtmikroskopie

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

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

Adenovirus-vermittelte genetische Entfernung von Signalmolekülen in kultivierten primären embryonalen Maus-Fibroblasten

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

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 ...

Isolation and Primary Culture of Rat Hepatic Cells

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

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

Isolierung und Primärkultur von Ratten Leberzellen

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

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

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

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

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

Imaging Lokale Ca<sup&gt; 2+</sup&gt; Signale in kultivierten Säugetierzellen

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

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

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

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

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

Erzeugen eines &quot;humanisierte&quot;<em&gt; Drosophila</em&gt; S2-Zelllinie Sensitive pharmakologische Hemmung der Kinesin-5

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

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

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

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

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

Glukoseaufnahme Messung und Response auf Insulin Stimulation in<em&gt; In vitro</em&gt; Kultivierte menschliche primäre Myotubes

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

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

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

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

Ganze Zelle Elektrophysiologie des primären kultivierten murinen Enterochromaffinen Zellen

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