Ringraziamo i membri del laboratorio Gelfand che hanno contribuito allo sviluppo di questi protocolli. Ricerca riportata in questa pubblicazione è stato sostenuto dal National Institute of General Medical Science del National Institutes of Health con il numero aggiudicazione R01GM052111 di VIG e basco Government Department of Education, dell&#39;Università e della Ricerca con il numero award BFI-2011-295 a UdC.

1. Cellule di Drosophila S2 <ol><li> Preparazione di vetrini ConA rivestiti <ol><li> Mettere coprioggetto in un rack ceramica e lavarli per immersione acido cromico per 1 ora. [ATTENZIONE: acido cromico è corrosivo e può causare irritazione di occhi, naso, gola e pelle]. </li><li> Sciacquare accuratamente con coprioggetto costantemente in esecuzione dH 2 O per 30 minuti fino a quando l&#39;acido è completamente lavato fuori. </li><li> Lasciare asciugare all&#39;aria coprioggetto e cappotto con ConA (soluzione 0,5 mg / ml di dH 2 O) per 30 minuti. </li><li> Sciacquare coprioggetto con dH 2 O per 15 minuti e lasciarli asciugare all&#39;aria. Coprioggetto ConA rivestiti possono essere memorizzati fino a 1 mese. </li></ol></li><li> Placcatura le cellule <ol><li> Consentono alle cellule S2 di crescere in modo esponenziale T25 o T75 cm 2 palloni a seconda del numero di cellule necessario per l&#39;esperimento. </li><li> Contare la densità cellulare utilizzando un emocitometro. Aggiungere 1 ml di crescita medium (per esempio Insect-Xpress) in un piatto di 35 millimetri di tessuto cultura con coprioggetto ConA rivestite, e delicatamente trasferire ~ 1 x 10 5 cellule al piatto. Questa densità cellulare è ottimale per l&#39;imaging perché i processi di celle diverse non si sovrappongano. </li><li> Al fine di indurre la formazione di processi, subito dopo la placcatura aggiungi medio 1ml con 5 mM CytoD al piatto (alla concentrazione finale di 2,5 mM CytoD). Consentire il completo sviluppo di processi incubando le cellule per almeno 2 ore a 25 ° C prima di imaging. </li></ol></li></ol> 2. Drosophila primaria Neuron Cultura <ol><li> Preparazione per la cultura neurone <ol><li> Preparare Drosophila mezzo di crescita neuronale (media di Schneider supplementato con siero: 20% fetale bovino, inattivati ​​termicamente a 55 ° C per 30 min; 5 mcg / insulina ml; 100 pg / ml di penicillina, 100 mg / streptomicina ml; 10 mcg / tetraciclina ml) . Questo supmezzo di pre realizzate Schneider può essere conservato a 4 ° C per 6 mesi. </li><li> Preparare fiale succo di mela agar per la raccolta di Drosophila embrioni (22,5 g di agar, 12,0 g di saccarosio e 750 ml di H 2 O in un pallone da 2 litri, autoclave per 20-25 minuti, aggiungere 250 ml di succo di mela e versare ~ 10 ml in ogni Drosophila fiala, dopo la solidificazione, collegare fiale con batuffoli di cotone, e avvolgere fiale con pellicola di plastica per alimenti per evitare di essiccazione del cibo). Il succo di mela in flaconcini di agar può essere conservato a 4 ° C in sacchetti di plastica ben chiusi per 4 settimane. </li><li> Cappotto lavata con acido 25 millimetri coprioggetto cerchio con ConA (vedi dettagli nel protocollo per le cellule di Drosophila S2) e sterilizzare sotto la luce UV per 10-15 min. Questi vetrini possono essere conservati per un mese. </li></ol></li><li> Drosophila cultura neuroni embrionali <ol><li> Mantenere giovane adulto vola in fiale di succo di mela agar addizionato con lievito secco per 1-2 giorni prima di raccogliere embrioni fo la preparazione neurone. </li><li> Il giorno di raccolta, al fine di sincronizzare gli embrioni raccolti, il trasferimento adulto vola a una mela fresco succo di agar fiala per 1 ora Precollection in luce e scartare questi embrioni. </li><li> Trasferimento vola a un nuovo flacone succo di mela agar, raccogliere embrioni per 1 ora, e trasferire le mosche per un altro flacone per un&#39;altra ora, sia nel buio per aumentare la resa embrione. </li><li> Rimuovere le mosche adulte dal succo di mela agar flacone e lasciare che gli embrioni si sviluppano altri 4 ore a 9-11 fasi a temperatura ambiente (4-6 embrioni hr-old sono tra palco 9-11). </li><li> Rimuovere gli embrioni spruzzando acqua in fiale e allentando gli embrioni dal succo di agar mela con un pennello piccolo (almeno 10 embrioni sono necessari per una buona neurone prep). </li><li> Trasferire gli embrioni a 100 micron maglia di nylon filtro cella con una pipetta di trasferimento per drenare l&#39;acqua (Prerisciacquare la pipetta di trasferimento in Drosophila tampone di lavaggio embrione, 0.4% NaCl, 0,03% TritonX-100). </li><li> Raccogliere gli embrioni dal filtro con un piccolo pennello, e trasferirli in una provetta da 1,5 ml riempita con 1 ml di Drosophila embrione di lavaggio (Nota: pulire le fibre di agar o di cotone utilizzando il pennello o puntali delle pipette). In questo protocollo, provetta da 1,5 ml dotato di un pellet usa e getta pestello di corrispondenza. </li><li> Lavare gli embrioni in Drosophila embrione lavaggio 3x. </li><li> Dechorionate gli embrioni in una soluzione fresca 1:01 di candeggina commerciale e il 95% di etanolo per 10 minuti [ATTENZIONE: candeggina può causare irritazione a occhi, naso, gola e pelle]. </li><li> Passare alla cappa coltura tissutale, e lavare gli embrioni 2x integrato nel mezzo di Schneider. </li><li> Lascia 200-300 microlitri di media nel tubo con gli embrioni, e delicatamente dissociare meccanicamente embrioni dechorionated utilizzando Kimble Chase pestelli pellet usa e getta che sono stati sterilizzati del 70% di etanolo (macinare 10-15x). </li><li> Centrifugare la miscela omogeneizzata a 20 xg per 2 minuti, e TRAnsfer il surnatante in una nuova provetta. [Questo passaggio è opzionale, aiuta a rimuovere i detriti embrione] </li><li> Centrifugare il surnatante a 550 xg per 2 minuti a pellet cellule. </li><li> Risospendere il pellet in 500 ml integrati cellule medie e di spin di Schneider giù di nuovo a 550 xg per 2 minuti, ripetere il ciclo di risospensione filatura volta. </li><li> Infine risospendere il pellet in ~ 100 ml, completata medio di Schneider e piastra le cellule su un vetrino ConA rivestito 25 millimetri in una sterile 35 millimetri piastra di Petri. Incubare per 10 min per permettere l&#39;adesione delle cellule. </li><li> Aggiungere 2 ml di terreno integrato di Schneider, con 5 micron CytoD per immergere il vetrino con le cellule attaccate. </li><li> Incubare il piatto in un umidificata 25 ° C incubatore notte prima di imaging. </li></ol></li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

Qui vi presentiamo protocolli dettagliati per studiare il trasporto di merci microtubuli-dipendente in cellule di Drosophila S2 e la cultura neurone primario. Entrambi i processi S2 e neuriti sono strutture riempito dal bundle microtubuli matrici che fungono da binari per il trasporto degli organelli. Due strategie sono tipicamente utilizzati per organelli etichetta: trasfezione di vettori che codificano una proteina fluorescente con una sequenza-organello il targeting o la colorazi...

Cellule di Drosophila S2 hanno guadagnato crescente popolarità per studiare molti processi cellulari. Queste cellule sono stati originariamente ottenuti da trypsinized embrioni fase tardiva della OregonR Drosophila melanogaster e mantengono le caratteristiche macrofagi-like 1. Cellule di Drosophila S2 offrono molti vantaggi rispetto ad altre linee cellulari. Essi sono coltivate a 25 ° C senza CO 2, e possono essere esposte per molte ore a temperatura ambiente senza la necessità di riscaldamento o scambio di gas. Ancora più importante, le cellule di Drosophila S2 sono molto sensibili al trattamento RNAi permettendo alta efficienza knockdown di proteine ​​di interesse. Cellule S2 sono altamente transfectable e linee cellulari stabili possono essere selezionati in meno di quattro settimane per permettere l&#39;espressione ectopica stabile di proteine ​​di interesse. Cellule S2 normalmente crescono sia liberamente associate, ma non si diffondono su un pallone o in sospensione. Essi possono essere indotti a diffondersi su vetrini rivestiti con una conca impianto lectinanavalin A (ConA). La periferia delle cellule spread è particolarmente sottile e quindi ideale per microscopia cellule vive. Protocolli dettagliati per coltura cellule S2, trattamento RNAi, imaging luce diretta e immunostaining possono essere trovati nel 2,3 letteratura. Il nostro laboratorio ha adottato le cellule S2 come sistema modello per studiare il trasporto merci basato microtubuli. Cellule di Drosophila S2 trattate con farmaci che depolimerizza o recide F-actina, come citocalasina D (CytoD), crescono lunghi processi pieni di microtubuli uniformemente polarizzati 4 -10, che lo rende un sistema ideale per studiare il movimento del carico lungo array microtubuli. Diversi parametri di trasporto in questi processi. (Cioè traiettorie lunghezze, velocità, direzionalità ecc) possono essere facilmente misurate utilizzando software particella di monitoraggio automatico, DiaTrack (Semasopht: http://www.microscopy.info/Organization/Details/4423; Dr . Pascal Vallotton: http://www.csiro.au/en/Organisation-Structure/Divisions/Mathematics-Informatics-and-Statistics/PascalVallotton.aspx # a4). Queste caratteristiche rendono le cellule S2 di un sistema interessante per studiare il trasporto di merci lungo i microtubuli in cellule di coltura di tessuti. Esperienze pilota in cellule S2 può essere esteso ad un modello fisiologicamente importante di studi trasporto merci in Drosophila neuroni primari. Simili alle cellule S2, colture di neuroni da embrioni dissociate sono permeabili a piccole molecole come coloranti e inibitori, e immagini ad alta risoluzione in tempo reale di trasporto organello possono essere eseguite in neuroni a temperatura ambiente. Utilizzo di Drosophila con differenti background genetico, possiamo esaminare la funzione dei geni nei neuroni primari per KO (mutazioni genetiche perdita-di-funzione), knockdown (dsRNA iniezione o espressione) o espressione ectopica (mosche transgeniche). In particolare, la frammentazione dei filamenti di actina mediante trattamento CytoD non impedisce crescita dei neuriti, invece crescono più velocemente, e quindi possiamo studiare microtubuli dependent trasporto organello nei neuroni CytoD trattati senza l&#39;influenza di actina Filamenti 11. Pertanto, colture neuronali primarie uniscono i vantaggi delle cellule di coltura di tessuti e volano genetica, che lo rende un ottimo sistema per studiare il trasporto di merci in un sistema fisiologico rilevante 10,12. Dal momento che diverse malattie neurodegenerative familiari potrebbero essere causati da o associate a difetti di trasporto merci (ad esempio, malattia di Parkinson 13, la malattia di Huntington 14, Morbo di Alzheimer 15,16, la Sclerosi Laterale Amiotrofica / ALS 17, HMN7B e Perry sindrome 18,19, e atassia spinocerebellare di tipo 5/SCA5 20), colture neuronali primarie possono essere utili per l&#39;analisi degli effetti di mutazioni specifiche che causano queste malattie sul trasporto microtubuli-dipendente. Infine, importanti proprietà di trasporto microtubuli-dipendente sono ben conservati tra i mammiferi e le mosche, ma <em&gt; Cultura genetica e delle cellule di Drosophila è meno costoso e richiede molto tempo, giustificando l&#39;uso di cellule in coltura di Drosophila per studiare aspetti essenziali del trasporto degli organelli in condizioni normali e patologiche.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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.

Cellule di Drosophila S2 placcato su un vetrino in presenza di lunghi processi ramificati forma farmaco-actina depolimerizzante pieni di microtubuli uniformemente polarizzati. Organelli si muovono lungo questi processi da motori microtubuli. Facilità di manutenzione, elevata sensibilità alla proteina RNAi mediato knock-down e procedure efficienti per la creazione di linee cellulari stabili rendere le cellule di Drosophila S2 un sistema modello ideale per studiare il trasporto merci con immagini dal vivo. I risultati ottenuti con le cellule S2 possono essere ulteriormente applicate a un sistema più fisiologicamente rilevanti: trasporto assonale nei neuroni primari in coltura da embrioni dissociate Drosophila. Neuroni in coltura crescono lunghi neuriti piene di microtubuli in bundle, molto simili ai processi S2. Come in cellule S2, organelli in neuroni in coltura possono essere visualizzate da entrambi i coloranti fluorescenti specifici per organelli o utilizzando marcatori fluorescenti organelli codificate dal DNA iniettato in embrioni precoci o espresse in transgenderic vola. Pertanto, il trasporto degli organelli può essere facilmente registrato in neuroni coltivati ​​su vetrini utilizzando immagini viventi. Qui si descrivono le procedure per la coltura e la visualizzazione di trasporto merci in cellule di Drosophila S2 e neuroni primari. Noi crediamo che questi protocolli rendono entrambi i sistemi accessibili per i laboratori che studiano il trasporto merci.

Cellule di Drosophila S2 attaccate sulla diffusione vetrino ConA rivestite in un piatto rotondo di forma &quot;pancake&quot; (Figure 1A e 1C). Tuttavia, quando le cellule S2 piastrate su vetrini in presenza di CytoD e propagate per 2-3 ore, formano processi sottili lunghi riempito dal microtubuli parallele (Figure 1B e 1D). Questi microtubuli hanno polarità uniforme con più-termina fuori 7. Time-lapse imaging di fluorescenza di <em...

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.

Cellule di Drosophila S2 e neuroni in coltura sono grandi sistemi per l&#39;imaging dei trasporti organello motorizzata in vivo. Qui si descrive protocolli dettagliati per la coltura di entrambi i tipi di cellule, la loro rappresentazione e analisi di trasporto.

Organelle Trasporti in Colta<em&gt; Drosophila</em&gt; Celle: Cell Line S2 e Primaria neuroni.

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 Views.  Feinberg School of Medicine, Northwestern University.  Cellule di Drosophila S2 e neuroni in coltura sono grandi sistemi per l'imaging dei trasporti organello motorizzata in vivo. Qui si descrive protocolli dettagliati per la coltura di entrambi i tipi di cellule, la loro rappresentazione e analisi di trasporto. 

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

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

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

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

Culture primarie neuronale dal cervello di tardo fase 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 ...

Ricerca

Biologia

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.

Immagini dal vivo di Dense-core Vescicole nella Primaria neuroni coltivati ​​ippocampale

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.

Preparazione del Drosophila Cellule S2 per la microscopia ottica

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-mediata rimozione genetica di molecole di segnalazione in fibroblasti di topo coltivate primarie embrionali

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.

Colture cellulari primarie da Drosophila Gastrula embrioni

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%.

Isolamento e la coltura primaria di cellule epatiche di ratto

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 locale Ca<sup&gt; 2+</sup&gt; Segnali in cellule di mammifero coltivate

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.

Generazione di un &quot;umanizzato&quot;<em&gt; Drosophila</em&gt; S2 Cell Line Sensibile alla farmacologica L&#39;inibizione di 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.

Misurazione e risposta alla somministrazione di glucosio alla stimolazione dell&#39;insulina in<em&gt; In vitro</em&gt; Myotubes primario umano coltivato

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.

A cellula intera elettrofisiologia di cellule enterocromaffini murino coltura primaria

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