Estamos em dívida para com os pacientes para a sua participação na coleção de tecidos para estas experiências. Um prêmio de academia do DoD OCRP ovário câncer (W81XWH-15-0253) e um piloto prêmio da Fundação de sonho de Colleen para Anirban K. Mitra apoiaram esta pesquisa.

O protocolo segue as diretrizes do Conselho regulamentação institucional da Indiana University.
1. preparação da pilha
<ol><li>
Isolamento e cultura de células mesoteliais primárias de
	<ol>
<li>Isolado primárias mesoteliais células humanas (HPMCs) de omento humana como descrito anteriormente,17,18 e cultivá-las em completar o meio de crescimento [modificado águia de Dulbecco médio (DMEM) contendo 10% de soro bovino fetal, 1% penicilina-estreptomicina, 1% não aminoácidos essenciais e vitaminas 1%] a 37 ° C e 5% de CO2. Nota: Os HPMCs são normalmente cultivadas para a confluência de 100% (figura 1A).</li>
	</ol>
</li>
	<li>
Condições de crescimento de células de câncer de ovário
	<ol>
<li>Desenvolvem-se células de câncer de ovário HeyA8 meio de crescimento completa em 37 ° C e 5% de CO2. Nota: Uso HeyA8 células cultivadas para cerca de 80% confluência (figura 1B).</li>
	</ol>
</li>
</ol>2. cultura de pilha
<ol><li>
Set-up cultura proximal
	<ol>
<li>Uso Transwell insere para placas boas 6 com uma membrana de policarbonato μm de espessura de tecido-cultura-tratados 10 com 0,4 μm poros (108 poros/cm2) e uma área de crescimento 4,67 cm2 . Se necessário, otimize para inserção de diferentes tamanhos, dimensionando o número de células semeado de acordo com a área de superfície de crescimento. Meio quente completo crescimento, tripsina e fosfato salino (PBS) a 37 ° C antes do uso.</li>
	</ol>
</li>
	<li>
Preparando as inserções
	<ol>
<li>Para propagar as células HeyA8 na superfície da membrana da inserção inferior, remova o encaixe de sua embalagem utilizando Pinças esterilizadas e coloque-invertida em um prato de cultura estéril de 15 cm.</li>
		<li>Use um prato de 15 cm, pois tem a profundidade para acomodar a inserção invertida, ou substituí-lo com qualquer recipiente estéril apropriado. Dependendo do experimento, coloque o número necessário de inserções no prato 15 cm.</li>
		<li>Rotule os três controles e três condições experimentais em conformidade com um marcador na borda das pastilhas.</li>
	</ol>
</li>
	<li>
Preparando as células cancerosas
	<ol>
<li>Para trypsinize as HeyA8 células cultivadas na confluência de 80% em um balão de2 25 cm (~ 2 x 106 células), adicionar 1 mL de tripsina (0,25%) para 40-60 s e neutralizar a tripsina com 6 mL de meio de cultura completo.</li>
		<li>Centrifugar as células a 500 x g , à temperatura ambiente (RT) por 3 min, desprezar o sobrenadante e ressuspender as células em 5 mL de meio de cultura completo. Conte as células ao vivo usando o método de exclusão do corante azul de Tripan.</li>
	</ol>
</li>
	<li>
Semeando as células cancerosas
	<ol>
<li>Suspenda a 100.000 células/mL de células de HeyA8 ao vivo no meio de crescimento completo. Cuidadosamente semente 80.000 HeyA8 células suspendidas em 800 μL de meio de cultura completo na parte inferior da inserção (que está agora virada para cima, uma vez que está invertida).</li>
		<li>As sementes das células para formar uma forma de cúpula, como assim as células permanecem na pastilha (Figura 1). Iniciar a partir do centro da membrana e mover-se para fora em círculos concêntricos ao Pipetar lentamente. Evite passar mais de 3 mm da borda para prevenir qualquer derramamento de médio porte. Nota: O número de células semeadas dependerá da taxa de crescimento das células e a duração do experimento. O número de células HeyA8 semeado para esta cultura proximal foram otimizado e as células será confluente 80 – 90% no final do terceiro dia.</li>
	</ol>
</li>
	<li>
Acessório de células de câncer
	<ol>
<li>Cubra o prato de 15 cm e cuidadosamente mover o prato contendo as inserções para a incubadora de CO2 . É fundamental para que não perturbe a gota de médio e células na pastilha nesta etapa. Embora as células a 37 ° C por 4 h permitir que as células cancerosas anexar para a inserção.</li>
	</ol>
</li>
	<li>
Preparando as células mesoteliais
	<ol>
<li>Após cerca de 4 h, trypsinize as HPMCs crescidos na confluência de 100% em um balão de2 75cm (~ 4 x 106 células) com 2 mL de tripsina (0,25%) por 1 – 2 min e neutralizar a tripsina com 12 mL de meio de crescimento completo.</li>
		<li>Centrifugar as células a 500 x g em RT por 3 min, Ressuspender as células em 10 mL de meio de cultura completo e contagem de células vivas, usando o método de exclusão do corante azul de Tripan.</li>
	</ol>
</li>
	<li>
Semeando as células mesoteliais
	<ol>
<li>Suspenda a 300.000 células/mL de HPMCs ao vivo no meio de crescimento completo. Adicione 2,5 mL de meio fresco completo de crescimento para cada poço de uma placa de 6. Cuidadosamente trazer o prato de 15 cm, contendo as inserções para fora para o bairro de biossegurança. Usando pinça estéril, a inserção da aleta e colocá-lo no poço da placa de 6-bem para que fique na posição vertical, com as células de HeyA8 ligadas à superfície inferior imergida no meio de crescimento completo (Figura 1).</li>
		<li>Semente de 450.000 HPMCs suspendidos em 1,5 mL do meio de crescimento no interior dos implantes com as células de HeyA8 ligadas à superfície, virado para o fundo do poço (Figura 1) ou para inserções sem quaisquer células HeyA8 para os controles HPMC. Adicione 1,5 mL de meio de crescimento sem pilhas para os controles HeyA8.</li>
	</ol>
</li>
	<li>
Crescer a cultura proximal
	<ol>
<li>Deixe a cultura proximal crescem a incubadora de CO2 a 37 ° C e 5% de CO2 por 72 h. Se necessário, o meio pode ser reposto da seguinte forma.<ol>
<li>Para o interior da inserção, remover 750 µ l e adicionar 750 µ l de meio fresco crescimento completo.</li>
			<li>Para o exterior da inserção (a 6 placa), retire 1 mL e adicionar 1 mL de meio fresco crescimento completo. O 1 mL foi escolhido para a conveniência de mudar o meio em uma única etapa. Se desejado, 1,25 mL pode ser removido e substituído em vez disso.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Recolha as células
	<ol>
<li>Recolha as células após 72 h da cultura proximal. Tome especial cuidado nesta etapa para evitar a contaminação cruzada de células HeyA8 e HPMCs. Trypsinize as células, centrifugue-los e lyse a pelota em vez de Lise das células na membrana para evitar uma contaminação cruzada.</li>
	</ol>
</li>
	<li>
Trypsinizing as células
	<ol>
<li>Enxágue ambos os lados da inserção com PBS 1x.</li>
		<li>As inserções de transferência para um novo poço e adicionar a tripsina. Adicionar 0,5 mL de tripsina no interior da inserção e adicionar 2 mL de tripsina para a placa de 6 para trypsinize as células de câncer ligadas à superfície inferior da inserção. Incubar a placa por 2 min a 37 ° C.</li>
	</ol>
</li>
	<li>
Neutralizando a tripsina
	<ol>
<li>Adicione 1 mL de meio de cultura completo no interior da inserção para neutralizar a tripsina, Pipetar para cima e para baixo e depois levar as células e transferi-los para um tubo de recolha rotulado. Adicione um adicional 2 mL do meio de crescimento completo de uma neutralização completa. Tenha cuidado para não perfurar a membrana enquanto pipetagem para evitar uma contaminação cruzada das células.</li>
		<li>Para coletar as células na parte inferior da inserção, pipete a tripsina 2ml que foi adicionada na etapa 12.2 para a superfície inferior 2 – 3 x para que as células cair na parte inferior da placa 6-poços. Neutralizar a tripsina na parte inferior da placa com 6 mL de meio de crescimento e transferir o conteúdo da placa para um tubo de recolha rotulado.</li>
	</ol>
</li>
	<li>
Coletando e lise das células
	<ol>
<li>Centrifugar as células a 500 x g por 3 min e Aspire os meios de comunicação. Resuspenda e lyse as pelotas de célula com 0,7 mL de reagente de Lise fenol e guanidina tiocianato-baseada para a extração de RNA. Isole o RNA usando qualquer kit apropriado.</li>
	</ol>
</li>
</ol>3. reação em cadeia da polimerase em tempo real quantitativa
Nota: As etapas a seguir descrevem uma reação em cadeia da polimerase em tempo real quantitativa (qPCR) para estudar as mudanças de expressões do gene como resultado a cultura proximal.
<ol><li>Prepare o cDNA de amostras de RNA para qPCR. Usar qualquer apropriado transcriptase reversa com um buffer correspondente e primers aleatórios para garantir a transcrição reversa do mRNA todos (ver Tabela de materiais).</li>
	<li>Avaliar as alterações dos níveis de expressão de RNAm de fibronectina (FN1) e E-caderina (CDH1) e transformar o fator de crescimento beta 1 (TGFβ1) por qPCR com gliceraldeído 3-fosfato desidrogenase (GAPDH) como um controle interno. É possível usar padronizados ensaios de expressão de gene comercialmente disponíveis (ver Tabela de materiais) ou para projetar primers.</li>
	<li>Compare as HeyA8 células crescidas na cultura proximal com HPMCs com os controles HeyA8 e as HPMCs crescidos em cultura proximal com os controles HPMC.</li>
</ol>

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Os autores não têm nada para divulgar.

Compreender o mecanismo de parácrina e justácrina sinalização entre as células é essencial para o desenvolvimento de um melhor conhecimento do tecido normal homeostase e doença condições7,8. Parácrina a maioria dos estudos são conduzidos através da recolha de sinalização condicionado médio de uma célula tipo e usá-lo para tratar o outro tipo de célula. Este método tem uma vantagem em sua simplicidade inerente. No entanto, isso não com precisã...

O papel de produtivas interações recíprocas entre as células cancerosas e o microambiente do tumor na progressão do tumor foi bem estabelecido e tornou-se um grande foco de pesquisa em câncer biologia1. Instâncias similares de sinalização bidirecional são cruciais durante o ferida cura, respostas imunes, angiogênese, nichos de células-tronco e desenvolvimento2,3,4,5,6 , 7 , 8. um tema comum em todos estes processos biológicos é que células respondem de várias maneiras para sinais extracelulares de seu microambiente que determinam o destino de celular, fisiologia do tecido e progressão da doença. Portanto, o foco tornou-se cada vez mais em direção a desenvolver uma melhor compreensão dos mecanismos envolvidos em tais comunicações célula-célula. A maioria de tais interações envolvem parácrina ou justácrina sinalização entre as células. Sinalização parácrina envolve a secreção de fatores específicos sinalização por uma célula que são percebidas pelos receptores correspondentes na outra célula na vizinhança, desencadeando uma resposta dentro de9,10, Considerando que justácrina sinalização requer o contato direto entre os componentes celulares das duas células envolvidas11,12.
Essa sinalização é um componente crucial na homeostase do tecido, bem como o microambiente do tumor. As células cancerosas beneficiam parácrina e justácrina fatores de células do estroma do tumor, incluindo cancro associado fibroblastos (CAFs), células do sistema imunológico e adipócitos13,14,15,16. A sinalização pode ser mediada por fatores de crescimento, citocinas, quimiocinas, etc., enquanto a sinalização justácrina envolve justapostos ligantes e receptores como sinalização Notch, ou interações entre integrinas e seus respectivos parácrina proteínas da matriz extracelular. Nós demonstramos a importância das interações recíprocas entre as células de câncer de ovário e CAFs no14, de progressão e metástase tumor. Da mesma forma, as interações de células de câncer de ovário metástase com as células mesoteliais, cobrindo o local da metástase regulam chave microRNAs e fatores de transcrição em células cancerosas que promovem a colonização metastático17, 18.
A maioria dos estudos na sinalização parácrina envolvem o uso de um meio condicionado coletado de tipo de uma célula para tratar o segundo tipo de célula com. Enquanto esta abordagem tem sido amplamente utilizada, ele não Replica eficazmente os níveis de alta concentração localizados do fator secretado no microambiente da célula receptora. Também não consegue reproduzir a cinética do fluxo contínuo do fator secretado sendo produzido por uma célula e recebido pela célula vizinha. Parácrina é eficaz em distâncias curtas, como os fatores secretados são nas concentrações necessárias apenas nas imediações da célula de origem e tendem a difundir e diluir para fora à medida que aumenta a distância de sinalização. Esta alta concentração localizada do fator secretado é essencial para desencadear uma resposta na célula do receptor. Além disso, a resposta nas células destinatários também é dependente o equilíbrio dos fatores secretados recentemente e seu esgotamento contínuo através de degradação, vinculação e internalização no destinatários células e difusão longe da célula de origem. Meio condicionado pode ser concentrado a conta para as altas concentrações localizadas presentes no microambiente, mas que exatamente não pode replicar as concentrações exatas. Além disso, ele não pode imitar a cinética natural de produção e depleção do fator envolvido. Para mais precisão replicar sinalização parácrina e separá-lo de justácrina mecanismos de sinalização, criámos um método de cultura proximal romance, que envolve os tipos de duas células em qualquer superfície de uma membrana porosa a crescer. Os poros são pequenos o suficiente para evitar interações justácrina e ainda permitir a troca de fatores secretados em concentrações elevadas localizadas. Dessa forma, este sistema retém a cinética da produção e depleção dos fatores parácrina.

<table>
	<tbody>
		<tr>
			<td>24 mm Transwell permeable support with 0.4 &#181;m Pore Polycarbonate Membrane Insert</td>
			<td>Corning (Costar)</td>
			<td>3412</td>
			<td>&#8226; 10 &#181;m thick translucent polycarbonate membrane 
			&#8226; Treated for optimal cell attachment 
			&#8226; Packaged 6 inserts in a 6 well plate, 4 plates per case 
			&#8226; Membrane must be stained for cell visibility 
			&#8226; Sterilized by gamma radiation</td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning (Falcon)</td>
			<td>353046</td>
			<td>Flat Bottom, TC-treated, sterile, with Lid</td>
		</tr>
		<tr>
			<td>15 cm culture dish</td>
			<td>Corning (Falcon)</td>
			<td>353025</td>
			<td>Sterile, TC-treated Cell Culture Dish</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning (Cellgro)</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential amino acids</td>
			<td>Corning (Cellgro)</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamins</td>
			<td>Corning (Cellgro)</td>
			<td>25-020-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin, 2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipets</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>Biosafety level II cabinet</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>FN1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs01549976_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>TGFB1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs00998133_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CDH1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs01023895_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs99999905_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy mini RNA isolation Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>43-688-13</td>
			<td></td>
		</tr>
		<tr>
			<td>HeyA8 ovarian cancer cells</td>
			<td>Obtained from Ernst Lengyel Lab, University of Chicago</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946; Neutralizing Antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1835-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a proximal culture method to study paracrine signaling between cells

Interações intercelulares desempenham um papel importante em muitos processos biológicos, incluindo a progressão do tumor, respostas imunes, angiogênese e desenvolvimento. Parácrina ou sinalização justácrina Medeia tais interações. O uso de um meio condicionado e estudos coculture são os métodos mais comuns para discriminar entre estes dois tipos de interações. No entanto, o efeito de concentrações elevadas localizadas dos fatores secretados para o microambiente durante as interações parácrina não é instaurado com precisão por meio condicionado e, assim, pode levar a conclusões imprecisas. Para superar este problema, criámos um método de cultura proximal para estudar a sinalização parácrina. Os tipos de duas células são cultivados em qualquer superfície de uma membrana de policarbonato µm de espessura 10 com 0,4 µm de poros. Os poros permitem a troca de fatores secretados e, ao mesmo tempo, inibem a sinalização justácrina. As células podem ser coletadas e lysed no ponto de extremidade para determinar os efeitos da sinalização parácrina. Além de permitir para localizadas gradientes de concentração dos fatores secretados, este método é passível de experimentos que envolvem períodos prolongados de cultura, bem como o uso de inibidores. Enquanto nós usamos este método para estudar as interações entre as células de câncer de ovário e as células mesoteliais que encontram no local da metástase, pode ser adaptado para qualquer dois tipos de células aderentes aos pesquisadores estudar parácrina sinalização em vários campos, incluindo desenvolvimento, imunologia e microambiente do tumor.

Metástase de células de câncer de ovário encontram células mesoteliais no local da metástase dentro da cavidade peritoneal19. Produtivo parácrina e justácrina interações com a ajuda de células mesoteliais na indução de respostas adaptáveis nas células de câncer de ovário, que permitem sucesso metástase17,18,20,21 . Para ...

Watch this Scientific Journal Video about A Proximal Culture Method to Study Paracrine Signaling Between Cells at JoVE.com

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Parácrina e justácrina interacções celulares desempenham um papel importante em muitos processos biológicos, incluindo a progressão do tumor, respostas imunes, angiogênese e desenvolvimento. Aqui, um método de cultura proximal é usado para estudar a sinalização parácrina onde as concentrações localizadas dos fatores secretados são mantidas, evitando o contato direto do celular.

Um método de cultura Proximal para estudar a parácrina sinalização entre as células

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and ...

a-proximal-culture-method-to-study-paracrine-signaling-between-cells

Research

JoVE Journal

Biology

9.4K visualizações.  Indiana University School of Medicine. Parácrina e justácrina interacções celulares desempenham um papel importante em muitos processos biológicos, incluindo a progressão do tumor, respostas imunes, angiogênese e desenvolvimento. Aqui, um método de cultura proximal é usado para estudar a sinalização parácrina onde as concentrações localizadas dos fatores secretados são mantidas, evitando o contato direto do celular.

Vídeo: A Proximal Culture Method to Study Paracrine Signaling Between Cells

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biologia

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para a Cultura de Embriões início pintainho ex vivo (Nova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Um método de alto rendimento para estudar a morfologia global organela em S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Oi-C: um método para estudar a arquitetura tridimensional de Genomas.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Um sistema para a cultura de células epiteliais pigmentadas da íris para estudo da regeneração Lens em Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Um método de genotipagem baseados em PCR para distinguir entre variedades do tipo selvagem e Ornamental de Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Visualização dos Vascular Ca 2 + Sinalização Disparado por parácrina ROS Derivado

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Um fluxo de trabalho de imagem de alta de conteúdo para estudar Grb2 Complexos de Sinalização por clonagem de expressão

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Método de Cultura para o Rato coração embrionário

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

A estimulação mecânica induzida por cálcio Propagação de Ondas em monocamadas de células: O Exemplo de Bovinos endotélio corneano

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Um método de cultura Proximal para estudar a parácrina sinalização entre as células

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