Este trabalho é apoiado pelo NIH R01 GM47607, CA163296 e NSF 1334493 a CET. Agradecemos a membros do laboratório Turner para a leitura crítica do manuscrito. Todos os dados apresentados neste relatório foram reproduzidas com permissão a partir de 2014 11 Wormer et al..

1. Preparação de Durotaxis Chambers <ol><li> Para preparar uma placa de 6 poços de cultura de tecidos, tarar a balança com um tubo cónico de 50 ml. Pesar aproximadamente 10 g de solução de base de PDMS no tubo de 50 ml (a solução é bastante viscosa). </li><li> Para uma 90: 1 substrato (Conformidade de ~ 1 kPa), dividir o peso medido da solução de base de PDMS no tubo por 90 para determinar a quantidade correcta de agente reticulante solução necessário. Adicionar a quantidade calculada da solução de agente reticulante PDMS ao mesmo tubo. Exemplo: 10 g / 90 = 0,11 g. Adicionar 0,11 g da solução de agente de reticulação à solução PDMS base de PDMS. NOTA: A base e do agente reticulante soluções são ambos fornecidos no kit de PDMS. </li><li> Misturar vigorosamente a mistura de base / agente de reticulação de PDMS durante 5 min à temperatura ambiente usando uma pequena espátula. Nesta fase, a mistura conterá um grande número de bolhas de ar. </li><li> Centrifuga-se o substrato de PDMS numa centrífuga de bancada durante 5 minutos a 50 xg à temperatura ambiente para remover a bubbles. Se ainda houver bolhas após a 5 min, centrifugar novamente. </li><li> Pipete 1 mL de 90: 1 PDMS substrato em cada poço de cultura de tecidos de 6 cavidades tratadas placa. As bolhas de ar restantes presentes nos PDMS pode ser eliminado nesta fase por popping-los usando uma agulha 21 G. Permitir que o PDMS para espalhar durante 30 min no poço. </li><li> Ferva 12 milímetros de vidro # 1 lamelas em água destilada por 5 min. Repita duas vezes e armazenar as lamelas em água destilada. </li><li> Coloque um, secou-se a lamela em cada poço da placa de cultura de tecidos tocando suavemente um lado da lamela na solução, em seguida, deixar cair o PDMS lamela para o PDMS. À medida que a lamela se instala, o PDMS começará a invadir ao longo dos bordos da lamela, mas não se cobri-lo completamente. Isto vai gerar uma interface entre o PDMS e de vidro após a cura (ver Figura 1A, B). </li><li> Incubar a placa a 70 ° C num forno durante 16 horas para curar (Harden) do PDMS. Coloque o plate em uma capa de cultura de células e UV esterilizar durante 10 min. NOTA: É melhor fazer as placas dentro de alguns dias de uso. No entanto, as placas podem ser envoltos em parafilme sem qualquer tampão e armazenado a 4 ° C durante até 2 semanas, sem qualquer queda significativa na qualidade. </li></ol> 2. celular Galvanização NOTA: Se se estudar o efeito do knockdown mediada por siRNA, executar o knockdown utilizando as instruções do fabricante ou o protocolo optimizado para o tipo de célula de escolha. <ol><li> Revestimento durotaxis cada câmara com 1 ml de 10 ug / ml de fibronectina em PBS sem cálcio e magnésio durante 1 hora a 37 ° C. Alternativamente, a fibronectina pode ser aplicado no dia antes da análise por incubação das PDMS com 10 ug / ml de fibronectina em PBS durante 16 horas a 4 ° C. Certifique-se de toda a superfície é submersa na solução de fibronectina em PBS. </li><li> Prepare desnaturado termicamente 1% BSA. Pesar 0,5 g de BSA e dissolvê-lo em 50 ml de PBS.Filtro de esterilizar a solução através de um filtro de 0,22 um e de calor a 80 ° C durante 12 min. Nota: Preparar esta solução no dia antes da célula de galvanização e armazenar a 4 ° C. </li><li> Aspirar a solução de fibronectina e lavar 3 vezes com PBS. Adicionar 1 ml de BSA desnaturado por calor em PBS a cada poço e incubar durante 30 min à temperatura ambiente. </li><li> Trypsinize e contar as células de escolha, enquanto as câmaras durotaxis estão bloqueando com o calor desnaturado BSA. </li><li> Placa 1 x 10 5 células em um volume de 2 ml em cada poço da câmara durotaxis, usando os meios necessários para o tipo particular de célula de escolha. Permitir que as células aderir e espalhada sobre o substrato durante 4 horas numa incubadora humidificada a 37 ° C com 5% de CO 2. NOTA: As células U2OS são rotineiramente mantidas em DMEM com 10% de FBS, suplementado com 2 mM de L-glutamina, piruvato de sódio 1 mM, e 10 UI / ml de penicilina, 10 ug / ml de estreptomicina. NOTA: O número de células utilizado neste exemplo é optimizado paraU2OS células. Optimização para outros tipos de células pode não ser necessária. Esta densidade dá as células espaço suficiente para migrar sem interações significativas com outras células. </li></ol> Imagem 3. Em células vivas <ol><li> Execute imagens de células vivas em um microscópio invertido, usando contraste de fase com uma objetiva de 10X. O microscópio deve ser equipado com uma câmara humidificada ambiente fechado, permitindo o controlo de temperatura a 37 ° C e 5% de CO 2 durante o exame a longo prazo. </li><li> Depois que as células se espalharam por cerca de 3,5 horas, montar a placa na câmara de microscópio. Deixar a amostra (s) para equilibrar na câmara durante 30 min. </li><li> Configure automatizado, multi-ponto de visitação no microscópio, se disponível. Concentre-se na interface entre as 90: 1 PDMS ea lamela de vidro e escolher aponta para imagem em todo o interface com um média de 40 pontos por durotaxis câmara. Imagem das células a cada 10 minutos até 16 horas. NOTA: A região de interesse aparece como duas linhas, com a linha exterior, correspondente ao bordo da lamela e a linha interna correspondente à interface real entre o PDMS e a lamela. Ver Figura 1B. </li></ol> Análise 4. Dados <ol><li> Gerar uma planilha como na Tabela 1. </li><li> Contar o número de eventos de passagem a partir dos PDMS à superfície do vidro e vice-versa de cada filme gerado. Grave o número de eventos de passagem na coluna apropriada na planilha excel. NOTA: Um evento de cruzamento é definido como o núcleo da célula, passando através da fronteira entre o PDMS e de vidro em um ou outro sentido. </li><li> Para quantificar múltiplos cruzamentos, contar o número de vezes que a célula atravessada a interface. Este número deve ser gravado na coluna Excel correspondente ao substrato sobre o qual a célula foi localizado no final do filme. Repita a análise para cada célula que atravessa a interface em tfilme que ele. Excluir células que migram para fora do campo de visão durante o exame. NOTA: É também importante para verificar, por contagem de células no início da experiência, as células que são capazes de aderir igualmente aos PDMS revestido com fibronectina e superfícies de lamela de vidro. </li><li> Calcula-se a percentagem de células que migraram a partir de PDMS para a superfície do vidro (isto é, submetidos a durotaxis). Adicione o número de cruzar eventos de suave a difícil e os múltiplos eventos de passagem, que terminou no disco e dividir pelo número total de eventos de passagem. </li><li> Calcula-se a percentagem de passagens múltiplas através da divisão do número de passagens múltiplas pelo número total de cruzamentos. </li></ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139 (5), 891-906 (2009).</li><li>Lo, C. M., Wang, H. B., Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+movement+is+guided+by+the+rigidity+of+the+substrate.">Cell movement is guided by the rigidity of the substrate.</a> Biophysical Journal. 79 (1), 144-152 (2000).</li><li>Plotnikov, S. V., Waterman, C. 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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+focal+adhesion+assembly:+a+close+relationship+studied+using+elastic+micropatterned+substrates.">Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.</a> Nature Cell Biology. 3 (5), 466-472 (2001).</li><li>Provenzano, P. P., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+signaling+through+the+cytoskeleton+regulates+cell+proliferation+by+coordinated+focal+adhesion+and+Rho+GTPase+signaling.">Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling.</a> Journal of Cell Science. 124 (8), 1195-1205 (2011).</li><li>Wang, Y. L., Pelham, R. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+focal+adhesion-localized+CdGAP+regulates+matrix+rigidity+sensing+and+durotaxis.">The focal adhesion-localized CdGAP regulates matrix rigidity sensing and durotaxis.</a> PloS ONE. 9 (3), e91815 (2014).</li><li>Trichet, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+large-scale+mechanosensing+mechanism+for+cellular+adaptation+to+substrate+stiffness.">Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness.</a> Proceedings of the National Academy of Sciences. 109 (18), 6933-6938 (2012).</li><li>Eyckmans, J., Boudou, T., Yu, X., Chen, C. 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The authors have no conflicts to disclose.

Aqui nós descrevemos um ensaio simples para estudar durotaxis em células migrando. Uma grande força deste ensaio é a facilidade de preparação das câmaras durotaxis utilizando PDMS. A rigidez dos substratos pode ser facilmente manipulado por alteração da proporção de solução de base de PDMS de agente de reticulação para permitir o estudo de vários elementos de rigidez no ensaio. No entanto, uma limitação potencial do sistema é que apenas as células são expostas a uma única mudança na rigidez do sub...

A matriz extracelular (ECM) é constituído por uma matriz complexa de proteínas estruturais e de reticulação, incluindo colagénio, fibronectina e laminina. Embora seja bem estabelecido que a ECM fornece um suporte estrutural importante para os tecidos celulares, há evidências crescentes de que indicam que as células respondem activamente a mudanças físicas no seu ambiente de ECM para regular processos celulares, incluindo diversas sobrevivência celular, diferenciação e migração da célula. Por exemplo, diferenças na rigidez do ECM podem conduzir as células estaminais mesenquimais para linhagens diferentes, com substratos macios (~ 1 kPa) a promoção de linhagens neurogénicos enquanto substratos rígidos (~ 25 kPa) promover a diferenciação osteogénica 1. Da mesma forma, foi demonstrado um aumento na rigidez da matriz estromal para promover a tumorigénese célula epitelial mamaria e invasão do tecido circundante 2,3. Um aspecto particularmente interessante deste mechanosignaling resultados da actividade em um processo conhecido como durotaxis, em que as células migram preferencialmente para um substrato mais rígido 4,5. Células sentir constantemente as características físicas do ambiente extracelular através da ligação do receptor de integrina ao ECM. Este, por sua vez, promove a acumulação de numerosas proteínas estruturais e de sinalização para os seus domínios citoplasmáticos para conduzir a formação de estruturas adesivas conhecidas como adesões focais ou contactos focais 6,7. Dado que as integrinas não têm actividade enzimática inerente, os sinais são transmitidos a partir do ECM através destas proteínas acessórias para coordenar a resposta da célula ao seu ambiente em mudança 8. Consequentemente, a identificação e caracterização das proteínas-chave envolvidas na regulação mechanosignaling durotaxis e é uma área importante de investigação. Vários sistemas de modelos têm sido desenvolvidos para estudar durotaxis in vitro, mas a maioria têmutilizados substratos de poliacrilamida revestidos com colagénio 4. No entanto, a preparação dos substratos de poliacrilamida pode ser tecnicamente exigente e o colagénio utilizado nestes ensaios devem ser quimicamente reticulado ao substrato 9. Polidimetilsiloxano (PDMS) substratos têm mostrado exibir propriedades mecânicas comparáveis ​​aos substratos de poliacrilamida a 10. No entanto, os substratos PDMS são preparadas simplesmente misturando uma proporção de base para o agente de reticulação e esses substratos podem ser revestidos com proteínas de ECM sem a necessidade de reticulação química, tornando assim mais fácil PDMS uma ferramenta para estudar os efeitos de rigidez no comportamento de células. Aqui, nós descrevemos como preparar uma câmara durotaxis simples em que um substrato macio PDMS é integrado com uma lamela de vidro rígida. O ensaio, conforme descrito abaixo, proporciona um método rápido e simples para estudar durotaxis. Para este estudo foram utilizadas células de osteossarcoma U2OS humanos, combinado com siRNA mediadaknockdown de cdGAP para estudar o papel desta proteína de adesão focal em durotaxis 11. É importante ressaltar que este protocolo pode ser facilmente adaptado às necessidades individuais. Outros tipos celulares podem ser substituídos para as células U2OS e qualquer proteína pode ser batido ou sobre-expresso para determinar os efeitos sobre o comportamento das células durante durotaxis. Além disso, este protocolo pode ser adaptado para incorporar proteínas fluorescente etiquetado para analisar sua dinâmica e comportamento usando FRAP ou FRET abordagens.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
		</tr>
		<tr>
			<td>#1 Cover glass 12mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-82</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Celltreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>15-017-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Fisher Scientific</td>
			<td>BP356-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>610077</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>21600-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Celltreat</td>
			<td>229456</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

As propriedades mecânicas da composição e da matriz extracelular são altamente variáveis ​​entre tipos de tecidos. Este tecido conjuntivo diversidade estroma grandemente impactos comportamento celular para regular os processos normais e patológicos, incluindo a proliferação celular, diferenciação, migração e adesão de sinalização direccional. A este respeito, a capacidade inata de determinados tipos de células para migrar em direcção um substrato mais rígido, ou menos complacente matriz é referida como durotaxis. Este fenómeno desempenha um papel importante durante o desenvolvimento embrionário, cicatrização de feridas e a invasão de células do cancro. Aqui, nós descrevemos um ensaio simples para estudar durotaxis, in vitro, utilizando PDMS (polidimetilsiloxano) substratos. Preparação do descrito durotaxis câmaras cria uma interface entre a rigidez do gel PDMS relativamente macio e uma lamela de vidro rígida. No exemplo fornecido, temos usado estes durotaxis câmaras para demonstrar um papel para o cdc42 / Rac1 GTPase ativando proteem, cdGAP, em mechanosensing e durotaxis regulamentação em células U2OS osteossarcoma humano. Este ensaio é facilmente adaptável a outros tipos e / ou knockdown de outras proteínas de interesse celulares para explorar seus respectivos papéis na mechanosignaling e durotaxis.

Um esquema da câmara durotaxis é mostrado na Figura 1A. Substrato PDMS macio (uma 90: 1 mistura de base de PDMS de reticulador soluções) é espalhada em uma 6 bem prato e uma lamela de vidro é colocado no topo do PDMS, que, em seguida, cobre parcialmente a superfície superior da lamela, criando, assim, uma interface entre os dois substratos de conformidade diferente. A rigidez do substrato macio PDMS é ~ 1 kPa, o que é comparável ao cumprimento típica do tecido do cérebro, en...

Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Um sistema simplificado para a Avaliação celular Mechanosensing e Durotaxis<em&gt; In Vitro</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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Universidad de Cartagena UDEC

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Biology

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

8.1K visualizações.  SUNY Upstate Medical University. Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Vídeo: A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

Transgénese mosaico Zebrafish de Avaliação Sequências Enhancer

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

Biologia

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivação eficiente dos recursos humanos Precursores cardíaca e Cardiomyocytes pluripotentes a partir de células estaminais embrionárias humanas com a indução de moléculas pequenas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de triagem para identificar fenótipos pós-embrionário em C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself Dispositivo de Recuperação de amostras criopreservadas cair acidentalmente em Tanques de armazenamento criogênico

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Na migração celular in vitro e invasão Ensaios

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

Ensaiando proteosomal Degradação em um sistema de livre-Cell em Plantas

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Um canal de gravação de patch-clamp anexado-Cell

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

A quantificação da Adesividade celular endotelial<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

Um sistema simplificado para a Avaliação celular Mechanosensing e Durotaxis

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Derivação eficiente dos recursos humanos Precursores cardíaca e Cardiomyocytes pluripotentes a partir de células estaminais embrionárias humanas com a indução de moléculas pequenas

RNAi de triagem para identificar fenótipos pós-embrionário em C. elegans

Do-It-Yourself Dispositivo de Recuperação de amostras criopreservadas cair acidentalmente em Tanques de armazenamento criogênico

Isolamento de Ribossomos ligados a polipeptídeos nascentes

Na migração celular in vitro e invasão Ensaios

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Um canal de gravação de patch-clamp anexado-Cell

A quantificação da Adesividade celular endotelial

Um protocolo para laboratório habitação de turquesa Killifish (Nothobranchius furzeri)