Agradecemos aos membros do laboratório Gelfand que contribuíram para o desenvolvimento destes protocolos. A pesquisa relatada nesta publicação foi apoiada pelo Instituto Nacional de Ciências Médicas Gerais dos Institutos Nacionais de Saúde sob o número prêmio R01GM052111 para VIG e pelo Departamento de Governo Basco da Educação, Universidades e Pesquisa sob o número prêmio BFI-2011-295 a UdC.

1. Células S2 de Drosophila <ol><li> Preparação de lamelas revestidas com ConA <ol><li> Coloque lamelas em um rack de cerâmica e lavá-los por imersão ácido crômico para 1 hora. [CUIDADO: o ácido crômico é corrosivo e pode causar irritação dos olhos, nariz, garganta e pele]. </li><li> Enxaguar abundantemente com lamelas funcionando constantemente dH 2 O por 30 minutos até que o ácido é completamente lavada. </li><li> Deixe o ar seco lamelas e revesti-los com ConA (solução de 0,5 mg / ml em dH 2 O) durante 30 min. </li><li> Lavar lamínulas com dH 2 O por 15 minutos e deixe-as secar naturalmente. ConA lamelas revestidas podem ser armazenadas até 1 mês. </li></ol></li><li> Plaqueamento das células <ol><li> Permitir que as células S2 a crescer exponencialmente em T25 ou T75 cm 2 frascos, dependendo do número de células necessário para o experimento. </li><li> Contagem da densidade de células utilizando um hemocitómetro. Adicionar 1 mL de crescimento médio (por exemplo Insecto-Xpress) de 35 mm no tecido do prato de cultura com ConA lamela revestida, e suavemente transferir ~ 1 x 10 5 células para o prato. Esta densidade de células é o ideal para imagens porque os processos a partir de células diferentes não se sobrepõem. </li><li> A fim de induzir a formação de processos, imediatamente após o plaqueamento adicionar meio de 1ml com 5 uM CytoD para o prato (a uma concentração final de 2,5 uM CytoD). Permitir o desenvolvimento completo dos processos por incubação das células, pelo menos durante 2 horas a 25 º C antes de imagem. </li></ol></li></ol> 2. Drosophila primária Cultura Neuron <ol><li> Preparação para a cultura neurônio <ol><li> Prepare Drosophila meio de crescimento neuronal (meio de Schneider suplementado com: 20% de soro fetal de bovino, inactivado por calor a 55 º C durante 30 min, 5 ug / ml de insulina, 100 ng / ml de penicilina, 100 ug / ml de estreptomicina, 10 ng / ml de tetraciclina) . Este supmeio de mentou Schneider pode ser armazenado a 4 º C por 6 meses. </li><li> Prepare frascos de ágar suco de maçã para a coleta de embriões de Drosophila (22,5 g de ágar, 12,0 g de sacarose e 750 ml de H 2 O em uma garrafa de 2 litros; autoclave para 20-25 min, adicione 250 ml de suco de maçã e despeje ~ 10 ml em cada Drosophila frasco, após a solidificação, ligue frascos com bolas de algodão, e embrulhe com filme frascos comida de plástico para evitar o excesso de secagem dos alimentos). O suco de maçã em frascos de ágar podem ser armazenados a 4 º C no bem-sacos plásticos fechados para 4 semanas. </li><li> Lavado com ácido Brasão 25 milímetros lamínulas círculo com ConA (ver detalhes no protocolo para células Drosophila S2) e esterilizá-los sob a luz UV por 10-15 min. Estas lamelas podem ser armazenadas por um mês. </li></ol></li><li> Drosophila cultura neurônio embrionário <ol><li> Mantenha jovem adulto voa em frascos de ágar suco de maçã suplementadas com levedura seca por 1-2 dias antes de coletar embriões fou preparação neurônio. </li><li> No dia em que a coleta, a fim de sincronizar os embriões colhidos, transferência adulto voa para uma maçã fresca suco frasco agar para uma pré-colheita de hr em luz e descartar esses embriões. </li><li> Transferência voa para um novo frasco de ágar suco de maçã, coletar embriões para 1 hora e transferir as moscas para outro frasco por mais uma hora, tanto no escuro para aumentar a produção de embriões. </li><li> Retire as moscas adultas do agar frasco de suco de maçã e deixar os embriões se desenvolvem mais 4 horas para estágios 9-11 à temperatura ambiente (4-6 embriões hr de idade estão entre fase 9-11). </li><li> Retire os embriões por esguichando água em frascos e soltando os embriões do ágar suco de maçã com um pincel pequeno (pelo menos 10 embriões são necessários para uma boa preparação neurônio). </li><li> Transferir os embriões para a 100 mm de malha de nylon filtro de células com uma pipeta de transferência para drenar a água (prerinse a pipeta de transferência em tampão de lavagem embrião Drosophila, 0,4% de NaCl, 0,03% TritonX-100). </li><li> Coletar os embriões do filtro usando um pequeno pincel, e transferi-los para um microtubo de 1,5 ml preenchida com 1 ml Drosophila lavagem embrião (Nota: limpar as fibras de algodão ou de ágar usando o pincel ou pontas de pipeta). Neste protocolo, a 1,5 ml microtubo vem com um pilão pellet descartável correspondente. </li><li> Lave embriões em lavagem embrião Drosophila 3x. </li><li> Dechorionate os embriões em uma nova solução 1:1 de água sanitária comercial e de etanol de 95% para 10 min [CUIDADO: água sanitária pode causar irritação nos olhos, nariz, garganta e pele]. </li><li> Mover-se para a capa de cultura de tecidos, e lavar 2x embriões em meio de Schneider suplementado. </li><li> Deixar 200-300 ul meio no tubo com os embriões, e suavemente dissociar mecanicamente embriões dechorionated usando Kimble perseguição pilões pelota descartáveis ​​que foram esterilizadas por etanol a 70% (moer 10-15x). </li><li> Centrifugar a mistura homogeneizada a 20 x g durante 2 min, e transfer o sobrenadante para um tubo fresco. [Este passo é opcional, que ajuda a remover os restos do embrião] </li><li> Centrifuga-se o sobrenadante a 550 g durante 2 min para sedimentar as células. </li><li> Ressuspender o sedimento em 500 ul de células suplementadas médio e de rotação de Schneider-se novamente a 550 x g durante 2 minutos, repetir o ciclo de re-suspensão de fiação uma vez. </li><li> Finalmente ressuspender o sedimento em ~ 100 ul suplementado meio de Schneider e prato das células numa lamela de 25 milímetros de ConA-revestido num estéril 35 milímetros placa de Petri. Incubar durante 10 min para permitir a fixação das células. </li><li> Adicionar 2 ml de meio de Schneider, suplementado com 5 uM CytoD para submergir a lamela com células ligadas. </li><li> Incubar o prato em um umidificado 25 ° C incubadora durante a noite antes de imagem. </li></ol></li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Aqui apresentamos protocolos detalhados para estudar o transporte de carga dependente de microtúbulos em células Drosophila S2 e cultura neurônio primário. Ambos os processos e neurites S2 são estruturas preenchidas por pacotes de microtúbulos matrizes que servem como pistas para o transporte de organelas. Duas estratégias são normalmente utilizados para organelas rótulo: transfecção de vetores que codificam uma proteína fluorescente com uma seqüência de metas organela...

Células Drosophila S2 ganharam popularidade crescente para estudar muitos processos celulares. Estas células foram originalmente obtidas a partir de embriões de fase tardia com tripsina de OregonR Drosophila melanogaster e manter as características de macrófagos como 1. Células S2 de Drosophila oferecem muitas vantagens em relação a outras linhas celulares. Elas são cultivadas a 25 ° C, sem CO 2, e podem ser visualizados por várias horas à temperatura ambiente, sem a necessidade de aquecimento ou de permuta de gás. Mais importante, as células de Drosophila S2 são muito sensíveis a um tratamento de alta eficiência, permitindo RNAi knockdown de proteínas de interesse. As células S2 são altamente transfectable e linhas celulares estáveis ​​pode ser seleccionada em menos de quatro semanas para permitir que a expressão ectópica estável de proteínas de interesse. As células S2 normalmente crescem ou fracamente ligado, mas não se espalhou sobre uma garrafa ou em suspensão. Eles podem ser induzidas para se espalhar em lamelas previamente revestidas com uma lectina de planta concanavalin A (ConA). A periferia das células de propagação é especialmente fina e, portanto, é ideal para a microscopia de células vivas. Os protocolos detalhados para a cultura de células S2, tratamento de RNAi, imagiologia de luz vivo e imunocoloração pode ser encontrada na literatura 2,3. O nosso laboratório adoptou células S2 como um sistema modelo para o estudo de transporte de carga à base de microtúbulos. Células S2 de Drosophila tratadas com qualquer droga que despolimeriza ou rompe F-actina, tais como a citocalasina D (CytoD), crescem processos longos cheios de microtúbulos uniformemente polarizadas 4 -10, o que o torna um sistema ideal para estudar a movimentação de cargas ao longo de matrizes de microtúbulos. Diferentes parâmetros de transporte nesses processos (ou seja. Trajetórias comprimentos, velocidades, direcionalidade, etc) pode ser facilmente medido usando o software de rastreamento de partículas automatizada, 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). Estas propriedades fazem células S2 um sistema atraente para o estudo de transporte de carga ao longo dos microtúbulos em células de cultura de tecidos. Experiências-piloto em células S2 pode ser estendido para um modelo fisiologicamente importante de estudos de transporte de carga em Drosophila neurônios primários. Semelhante para células S2, neurónios cultivados a partir de embriões dissociadas são permeáveis ​​a moléculas pequenas, tais como corantes e inibidores, e alta resolução de imagem ao vivo de transporte organela pode ser realizada em neurónios à temperatura ambiente. Usando Drosophila com diferentes origens genéticas, podemos examinar a função do gene em neurônios primários por nocaute (genéticos mutações de perda de função), knockdown (injeção dsRNA ou expressão) ou expressão ectópica (moscas transgênicas). Especificamente, a fragmentação dos filamentos de actina utilizando tratamento CytoD não impede crescimento de neuritos, em vez disso eles crescem mais rápido e, assim, podemos estudar microtúbulos-dependtransporte organela nt em neurônios tratados com CytoD sem a influência de filamentos de actina 11. Portanto, culturas neuronais primárias combinam as vantagens das células de cultura de tecidos e genética voar, o que o torna um grande sistema para estudar o transporte de carga em um sistema relevante fisiológica 10,12. Uma vez que várias doenças neurodegenerativas familiares poderiam ser causadas por ou associadas com os defeitos de transporte de carga (por exemplo, doença de Parkinson 13, doença de Huntington 14, a doença de Alzheimer 15,16, Esclerose Lateral Amiotrófica / ALS 17, HMN7B e Perry síndrome 18,19, e tipo de ataxia espinocerebelar 5/SCA5 20), as culturas neuronais primárias podem ser úteis na análise dos efeitos das mutações específicas que causam estas doenças em transporte dependente de microtúbulos. Finalmente, as propriedades importantes de transporte dependente de microtúbulos são bem conservada entre os mamíferos e as moscas, mas <em&gt; Cultura genética e células de Drosophila é menos caro e demorado, o que justifica o uso de células de Drosophila cultivadas para estudar aspectos essenciais de transporte organela em condições normais e patológicas.

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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.

As células S2 de Drosophila semeadas em uma lamela, na presença de quaisquer processos longos não ramificados formam droga-despolimerização da actina cheios com microtúbulos uniformemente polarizadas. Organelos mover ao longo destes processos pelos motores de microtúbulos. Fácil manutenção, de alta sensibilidade à proteína do RNAi mediada por knock-down e procedimento eficiente para a criação de linhas celulares estáveis ​​fazer células Drosophila S2 um sistema modelo ideal para estudar o transporte de carga por imagens ao vivo. Os resultados obtidos com as células S2 podem ainda ser aplicado a um sistema mais fisiologicamente relevante: o transporte axonal em neurónios primários cultivadas a partir de embriões de Drosophila dissociados. Neurônios cultivados crescer neurites longos cheios de microtúbulos empacotados, muito semelhantes aos processos S2. Como em células S2, organelas em neurônios cultivados pode ser visualizado por qualquer corantes fluorescentes específicos de organelas ou usando marcadores fluorescentes organelas codificadas pelo DNA injetado em embriões ou expressa em transgénicaic voa. Portanto, o transporte organela pode ser facilmente gravado em neurônios cultivados em lamínulas usando imagens vivas. Aqui nós descrevemos os procedimentos de cultura e visualizando o transporte de carga em células Drosophila S2 e neurônios primários. Acreditamos que estes protocolos fazer os dois sistemas acessíveis para os laboratórios que estudam o transporte de carga.

Células Drosophila S2 ligados na lamela propagação ConA revestido em uma rodada plana &quot;panqueca&quot; forma (Figuras 1A e 1C). No entanto, quando as células S2 semeadas em lamelas na presença de CytoD e deixada espalhar-se para 2-3 horas, formam processos longos e finos carregados em microtúbulos paralelas (Figuras 1B e 1D). Estes microtúbulos têm polaridade uniforme com plus-termina a 7. Time-lapse imagens de fluorescê...

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.

Células Drosophila S2 e neurônios cultivados são grandes sistemas para a imagem latente de transporte organela motorizado in vivo. Aqui nós descrevemos protocolos detalhados para o cultivo de ambos os tipos de células, a sua imagem e análise de transporte.

Transporte Organelle em Cultivadas<em&gt; Drosophila</em&gt; Células: Linha celular S2 e primário neurônios.

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 visualizações.  Feinberg School of Medicine, Northwestern University.  Células Drosophila S2 e neurônios cultivados são grandes sistemas para a imagem latente de transporte organela motorizado in vivo. Aqui nós descrevemos protocolos detalhados para o cultivo de ambos os tipos de células, a sua imagem e análise de transporte. 

Vídeo: 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.

Culturas primárias neuronais do cérebro de Tarde Stage Drosophila pupas

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

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.

Imagens ao vivo de vesículas com núcleo denso na Primária Cultivadas neurônios do hipocampo

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.

Preparação de Drosophila Células S2 para microscopia de luz

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.

Adenovírus mediada por remoção genética das moléculas de sinalização em fibroblastos de rato cultivadas Primária embrionárias

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.

Culturas de células primárias de Drosophila Os embriões Gástrula

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 cultura primária de células hepáticas de ratos

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.

Imagem local Ca<sup&gt; 2+</sup&gt; Sinais em células de mamíferos em cultura

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.

Gerando um &quot;humanizado&quot;<em&gt; Drosophila</em&gt; S2 linha de células sensíveis a inibição farmacológica de cinesina-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.

Medição da captação de glicose e resposta à estimulação de insulina em miotubos primários humanos cultivados in vitro

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.

Eletrofisiologia celular de células enterocromafins murino culta primário

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