Trabalho é o nosso laboratório é suportada por concessões do National Institutes of Health.

Preparação <ul><li> Lamínulas devem ser autoclavados. </li><li> Água destilada ou deionizada estéril deve ser utilizada para preparar soluções e lamínulas para lavar roupa. </li><li> AC (40% w / v) e bis-AC (1% w / v) soluções são esterilizados por filtração 0,2 mM. Prepare persulfato de amônio 10% (APS; água 100μg/ml), pouco antes de usar e filtro estéril. Substituir a solução APS mensal. </li><li> Reagentes químicos, tais como 3-APTMS, clorofórmio, glutaradehyde, NHS, e SurfaSil que não podem ser autoclavados são mantidos em uma garrafa atribuído utilizados exclusivamente para a preparação de hidrogéis. </li><li> Para melhores resultados, hidrogéis deve ser utilizada dentro de um par de dias após a incubação durante a noite com a proteína ECM apropriado. </li><li> Prepare uma solução SurfaSil 10% em clorofórmio (10 ml é geralmente suficiente para 20 lamínulas superior) em um tubo de polipropileno de 50 ml Falcon antes da efusão do hidrogel. (Nosso laboratório normalmente siliconizes as lamelas top durante a etapa de 0,5% de glutaraldeído de incubação). Adicionar as lamelas ao tubo Falcon e rock durante pelo menos 10 min. Decantar a solução SurfaSil e ar seco sobre as lamelas Kimwipes no gabinete de segurança biológica, onde os hidrogéis será preparado. </li><li> Prepare a inativado pelo calor solução BSA da seguinte forma: a 20 mg / ml solução de ácidos graxos livres BSA em PBS é incubada em banho-maria de 68 ° C por 30 min. A solução é, então, estéril filtrado e armazenado a 4 ° C. </li></ul> Procedimento <ol><li> Coloque uma camada de Parafilm na metade inferior de uma placa de Petri de 150 mm. </li><li> Lugar até 9 de 25 mm lamínulas em cima do Parafilm e cobri-los com 1 ml 0,1 M NaOH. Incubar por 3 min e em seguida, aspire com uma linha de vácuo. </li><li> Trabalhando em um capuz químico, coloque 0,5 ml de 3 APTMS em cada lamela. Incubar por 3 min e depois aspirar o APTMS. Se você esperar muito tempo, uma espuma irá se formar. </li><li> Lave as lamelas uma vez com 20 ml de água deionizada no mesmo prato. Retirar as lamelas do prato com a pinça curva e transferi-los, com seus tratados do lado voltado para cima, a um prato de 150 mm nova. Lavar as lamelas com água deionizada por três vezes, na cadeira de balanço, por 10 min cada lavagem. Se você não conseguir remover todos os APTMS, ele irá reagir com o glutaraldeído na próxima etapa e deixar um precipitado nublado branco (Figura 1). </li><li> Descongelar o glutaraldeído (~ 10 min antes do uso). </li><li> Utilizando uma pinça curva, as lamelas de transferência para um prato limpo camadas com Parafilm e aspirar todo o líquido restante. Use uma linha de vácuo ou Kimwipe para apagar o restante da água, conforme necessário. </li><li> Cobrir cada lamela completamente com 0,5 ml de glutaraldeído 0,5% em água deionizada estéril e incubar por 30 min em uma capa química. Aspirar o glutaraldeído. Lavar as lamelas como no passo 4. Seca as lamelas completamente. [Você pode parar por aqui e deixar as lamelas durante várias semanas em uma área seca]. </li><li> Quando você está pronto para preparar o hidrogéis, utilize uma pinça curva para transferir as lamelas, de cabeça reativa, para uma folha de Parafilm que foi gravado na superfície do gabinete de segurança biológica. Certifique-se que as lamelas são planas na superfície Parafilm. </li><li> Preparar uma solução saturada NHS em tolueno. (Dissolver uma pequena quantidade de NHS em tolueno suficiente para o experimento particular. Continue adicionando pouco a pouco NHS até que o NHS não mais se dissolve. A solução saturada é geralmente muito nublado e rosa.) </li><li> Em seguida, preparar a acrilamida, bis-acrilamida, água e APS para atingir o percentual desejado de acrilamida. Adicionar os reagentes em tubos de microcentrífuga, conforme descrito abaixo para um volume total de 0.8ml. </li><li> Então, uma alíquota de cada vez, adicione o SNS e para o Temed 0,8 ml solução AC, vortex brevemente e imediatamente despeje 05/03 géis utilizando 140 mL por lamela na capa de segurança biológica. Sempre que possível, todas as medidas a partir deste ponto deve ser realizado em uma cabine de segurança biológica. [Nota: lamelas de diferentes tamanhos pode ser utilizado com base na necessidade. Se lamínulas de 18 mm são utilizadas, use ~ 33 mL de solução AC por lamínula e um de 18 mm lamela superior siliconizado.] <table border="1"><tbody><tr><td></td><td colspan="4"> Bis-AC (%) </td></tr><tr><td></td><td> 0,3 (dura) </td><td> 0,15 </td><td> 0,06 </td><td> 0,03 (soft) </td></tr><tr><td></td><td> mL </td><td> mL </td><td> mL </td><td> mL </td></tr><tr><td> Água </td><td> 402 </td><td> 522 </td><td> 594 </td><td> 618 </td></tr><tr><td> CA </td><td> 150 </td><td> 150 </td><td> 150 </td><td> 150 </td></tr><tr><td> Bis-AC </td><td> 240 </td><td> 120 </td><td> 48 </td><td> 24 </td></tr><tr><td> APS </td><td> 8 </td><td> 8 </td><td> 8 </td><td> 8 </td></tr><tr><td> Temed</td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> NHS </td><td> 228 </td><td> 228 </td><td> 228 </td><td> 228 </td></tr></tbody></table></li><li> Rapidamente coloque a lamínula de 25 mm siliconizado em cima de cada gel antes que comece a polimerizar. Além da lamela superior deve permitir que a AC para cobrir completamente a lamela inferior. Incubar esse &quot;sanduíche&quot; em temperatura ambiente até que a AC polimeriza. (Verifique a solução AC residual no tubo de microcentrífuga para determinar quando ocorreu a polimerização. Normalmente alguns minutos para géis firmes e um pouco mais para os moles será suficiente.). </li><li> Com cuidado, pegue o sanduíche (luvas estéreis on!) E deslize a lamela superior até que pende sobre o gel polimerizado. Você pode, então, retire-o do gel. Se você esperar muito tempo antes de retirar a lamela superior, o gel vai rasgar como a lamela é removido. </li><li> Descartar a lamela superior. Coloque o fundo gel-lamelas (doravante denominado o hidrogel) em 6 bem-placas com 2 ml PBS / poço. PBS podem ser adicionados antes ou depois do gel lamela para cada poço. Lavar os hidrogéis com PBS três vezes, em uma cadeira de balanço, 5 min por lavagem. </li><li> Repita os passos 11-14 até o número necessário de hidrogéis foram preparados. </li><li> Cobrir cada hidrogel com 2 ml de uma solução de fibronectina (3 mg / ml em PBS) ou proteína ECM (ver Klein et al. 13 para detalhes.). A proteína ECM torna-se covalentemente ligado ao hidrogel durante a incubação overnight a 4 ° C. </li><li> Aspirar a solução de ECM e bloquear NHS não reagiu com 1mg/ml, inativado pelo calor BSA de ácidos graxos livres no soro livre de mídia por pelo menos 30 min a 37 ° C em uma cultura de células incubadora. Lavar a hidrogéis uma vez com PBS estéril ou meio de cultura celular. </li><li> Células placa no meio de cultura apropriado contendo FBS. O número de células inoculadas no hidrogel deve ser determinada pelo usuário com base no grau de disseminação de células e confluência necessária para a experimentação. Cerca de 10 5 células é geralmente suficiente para a análise de western blot e qPCR. </li><li> Após o período de incubação, a proteína celular ou mRNA pode ser extraído. As lamelas são cuidadosamente retirados dos poços usando uma pinça curva e colocado (célula voltada para baixo) em cima de 100 gotas mL de tampão de lise que foram espaçadas (2-3cm) ao longo de uma folha de Parafilm na bancada do laboratório. (Nós usamos padrão tampão de amostra SDS e TRIzol, respectivamente, para preparar amostras para blotting ocidental e para isolar RNA para qPCR). Incubar as células com o tampão de lise de exatamente um minuto. Retirar as lamelas e transferir o tampão de lise a um tubo de microcentrífuga. Alternativamente, para extração de RNA somente, o usuário pode transferir cada hidrogel para uma placa de seis poços novos e adicionar 1 ml de TRIzol / poço. Incubar por 3 minutos e retire solução TRIzol para armazenamento em um tubo de microcentrífuga. </li></ol> Informações adicionais sobre os procedimentos, tais como imunofluorescência, BrdU coloração, transfecção, etc para as células semeadas em hidrogéis é descrita em Klein et al. 2007 13. Resultados representante Lavagem completa das lamelas seguintes adição de APTMS é um passo importante na produção de &quot;reativa&quot; lamínulas. Se um falhar para remover o APTMS completamente, ele irá reagir com o glutaraldeído na etapa seguinte e produzir um precipitado branco cloudy como visto na Figura 1A. A Figura 1B mostra uma lamela adequadamente lavadas e secas. Se o precipitado se desenvolve, todo o procedimento deve ser reiniciado desde o início como a lamela não é mais utilizável. Depois da formação de hidrogel e revestimento com proteínas ECM durante a noite, as células podem ser semeados no dia seguinte. Como a Figura 2 mostra, há uma nítida diferença entre célula se espalhando em hidrogéis duro contra macio. Como pode ser visto por phalloidin coloração em fibroblastos do ratinho embrionárias (MEFs), células se espalham em maior medida na rigidez em comparação com hidrogéis macio. De fato, a maioria das células associados a um hidrogel macia permanecerá compacta e anexar menos eficiente. Embora a morfologia MEF só é mostrado na Figura 2, a diferença na célula se espalhando é consistente em várias linhagens de células outras testadas 11-12,14. Segredos para o Sucesso <ol><li> Uma vez que o procedimento é iniciado, ele é muito importante para manter as lamelas com o lado &quot;reativo&quot; up e ter em mente que lado foi revestida. </li><li> As luvas devem ser usadas todo tempo durante o procedimento para fornecer um ambiente de trabalho que é tão estéril quanto possível. </li><li> A maioria dos passos iniciais são realizadas entre uma Cobertura de vapores químicos ea bancada do laboratório. Todas as etapas subseqüentes para remover o excesso de glutaraldeído as lamelas deve ser executada sob um armário de segurança biológica. </li><li> Devido às diferenças na célula distding (Figura 2), recomendamos semear o dobro do número de células em hidrogéis suave em comparação com hidrogéis dura. </li><li> O processo de polimerização AC é extremamente rápido. Para usuários de primeira vez, pode-se diminuir a quantidade de APS ou Temed na solução para prolongar o processo de polimerização. Não tente se preparar mais do que uma poucas lamelas de uma vez. </li><li> Em estudos do ciclo celular em G0, onde a sincronização é necessária, nós geralmente soro fome células em placas de cultura de plástico, trypsinize as células e reseed-los em hidrogéis de conformidades diferentes na presença de mitógenos (FBS e / ou fatores de crescimento). Este procedimento garante que a população de partida da G0-sincronizado células é idêntico em todas as amostras. No entanto, para estudos de transdução de sinal de muitos, o período de estimulação necessária mitógeno pode não ser suficiente para permitir a fixação e disseminação de células. Neste caso, nós soro-fome as células na hidrogéis e depois diretamente estimulá-los com mitógeno. </li></ol><img src="/files/ftp_upload/2089/2089fig1a.jpg" alt="Figura 1a" /> Figura 1A. Inadequadamente lavado lamela. Após adição de APTMS, lamínula foi lavada por 1-2 minutos antes da adição da solução de glutaraldeído. Um precipitado formulários na lamela que não tenha sido lavados de acordo com as etapas descritas no procedimento. <img src="/files/ftp_upload/2089/2089fig1b.jpg" alt="Figura 1b" /> Figura 1B. Devidamente lavados lamela. Adição Após a APTMS, lamínula foi lavada por três vezes 10 minutos cada antes da adição da solução de glutaraldeído. Nenhum precipitado se forma na lamela. <img src="/files/ftp_upload/2089/2089fig2.jpg" alt="Figura 2" /> Figura 2. Morfologia das células em hidrogéis de diferentes rigidez. Fundada MEFs foram semeadas em fibronectina revestido hidrogéis de rigidez alta ou baixa para 9 horas. Após o período de incubação, as células foram fixadas, permeabilizadas e coradas com FITC-phalloidin que se liga ao f-actina. MEFs em gel de alta rigidez apresentam fibras de stress e são bem distribuídos em comparação com aqueles semeados em hidrogéis baixa rigidez. Barra de escala = 50 ìm. <img src="/files/ftp_upload/2089/2089fig3.jpg" alt="Figura 3" /> Figura 3. Representante quantitativa resultado da PCR D1 rato ciclina níveis de mRNA. Serum fibroblastos de rato faminto embrionárias foram semeadas em alta (3% de acrilamida) ou baixo hidrogéis (0,3% de acrilamida) rigidez e estimuladas com FBS 10% para 9 horas. Após extração de RNA, análise em tempo real PCR quantitativa foi realizada para os níveis de ciclina D1 mRNA (normalizados para 18S RNA). G0 representa mRNA ciclina D1 a partir de células quiescentes. Níveis de ciclina D1 mRNA aumentar significativamente em hidrogéis rígida, mas não em hidrogéis macio. Os dados são média + / - SD de reações duplicar PCR.

<ol>
	<li>Beattie, D., Xu, C., Vito, R., Glagov, S., Whang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+analysis+of+heterogeneous,+atherosclerotic+human+aorta.">Mechanical analysis of heterogeneous, atherosclerotic human aorta.</a> J Biomech Eng. 120, 602-607 (1998).</li><li>Bernini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness,+intima-media+thickness+and+carotid+artery+fibrosis+in+patients+with+primary+aldosteronism.">Arterial stiffness, intima-media thickness and carotid artery fibrosis in patients with primary aldosteronism.</a> J Hypertens. 26, 2399-2405 (2008).</li><li>Boonyasirinant, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+stiffness+is+increased+in+hypertrophic+cardiomyopathy+with+myocardial+fibrosis:+novel+insights+in+vascular+function+from+magnetic+resonance+imaging.">Aortic stiffness is increased in hypertrophic cardiomyopathy with myocardial fibrosis: novel insights in vascular function from magnetic resonance imaging.</a> J Am Coll Cardiol. 54, 255-2562 (2009).</li><li>Discher, D. E., Janmey, P., 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=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+substrate.">Tissue cells feel and respond to the stiffness of their substrate.</a> Science. 310, 1139-1143 (2005).</li><li>Duprez, D. A., Cohn, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness+as+a+risk+factor+for+coronary+atherosclerosis.">Arterial stiffness as a risk factor for coronary atherosclerosis.</a> Curr Atheroscler Rep. 9, 139-144 (2007).</li><li>Lee, R. 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Um elemento crucial do processo de polimerização hidrogel é evitar a formação de bolhas de ar que vai permitir que as células se ligam à lamela de vidro, em vez de o hidrogel ECM revestido em si. Isto pode ser evitado com cuidado pipetagem a solução de polimerização após vórtex e visualmente para que não se têm bolhas de ar ficam presas no gel. Recomendamos sempre a preparar adicionais &quot;reativa&quot; lamínulas e hidrogéis para garantir ter o suficiente para a experimentação. <p class="jove_co...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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		<tbody>
		
<tr>
<td>Glutaraldehyde, 70%</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7776</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>3-APTMS (3-Aminopropyltrimethosysilane 97%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>SurfaSil Siliconizing Fluid</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>42800</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>NHS (N-hydroxysucinimide Ester)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A-8060</td>
<td>Store at 4&deg;C Replace monthly</td>
</tr>
<tr>
<td>Albumin, bovine serum, essentially fatty acid free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6003-100G</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>Coverslips (25mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-86 25 Cir 1D</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (18mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-84 18 Cir 1D</td>
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studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels

Rigidez do tecido é um determinante importante da função celular, e mudanças na rigidez do tecido são comumente associados com câncer, fibrose e doenças cardiovasculares 1-11. Abordagens tradicionais de células biológicas para estudar a função celular envolvem a cultura de células em um substrato rígido (pratos de plástico ou de lamínulas de vidro) que não pode explicar o efeito de um ECM elástica ou as variações de ECM rigidez entre os tecidos. O modelo em condições de tecido vivo cumprimento in vitro, nós e os outros usam ECM-revestido hidrogéis. Em nosso laboratório, os hidrogéis são baseados em poliacrilamida, que podem imitar a faixa de tecido conformidades visto biologicamente 12. &quot;Reativa&quot; lamínulas são gerados por incubação com NaOH seguidos pela adição de 3-APTMS. Glutaraldeído é usado para cross-link 3-APTMS eo gel de poliacrilamida. A solução de acrilamida (AC), bis-acrilamida (Bis-AC) e persulfato de amônio é utilizado para a polimerização do hidrogel. N-hydroxysuccinimide (NHS) é incorporado a solução CA para crosslink proteína ECM para o hidrogel. Após a polimerização do hidrogel, a superfície do gel é revestido com uma proteína de ECM de escolha, tais como a fibronectina, vitronectina colágeno, etc A rigidez de um hidrogel pode ser determinada por reologia ou microscopia de força atômica (AFM) e ajustado pela variação do percentual de AC e / ou bis-AC na solução 12. Desta forma, a rigidez substrato pode ser combinada com a rigidez dos tecidos biológicos que também pode ser quantificada através de reologia ou AFM. As células podem ser semeados nesses hidrogéis e cultivadas com base nas condições experimentais necessárias. Imagem das células e sua recuperação para a análise molecular é simples. Para este artigo, definimos substratos moles como aqueles que têm módulos elásticos (E) &lt;3000 Pascal e substratos rígidos / tecidos como aqueles com E&gt; 20000 Pascal.

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Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

O efeito da rigidez substratos em função celular podem ser modelados<em&gt; In vitro</em&gt; Usando hidrogéis de poliacrilamida de diferentes conformidades.

Estudando os efeitos da rigidez Matrix em função celular usando acrilamida baseado Hidrogéis

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

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21.9K visualizações.  University of Pennsylvania. O efeito da rigidez substratos em função celular podem ser modelados In vitro Usando hidrogéis de poliacrilamida de diferentes conformidades.

Vídeo: Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

O encapsulamento celular em Hidrogéis 3D para Engenharia de Tecidos

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Biologia

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

Medir a rigidez à flexão de células bacterianas Usando uma armadilha óptica

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Identificar os efeitos das mutações BRCA1 em Recombinação Homóloga usando células que expressam BRCA1 tipo selvagem endógena

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Analisando a função de GTPases Pequenas pela microinjeção de Plasmids em Polarized Células Epiteliais

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Visualização da função respiratória mitocondrial usando Citocromo C Desidrogenase Oxidase / Succinato (COX / SDH) Histoquímica rotulagem dupla

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Criando ambientes definidos gasosos para estudar os efeitos da hipóxia na<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Medição dos Efeitos de bactérias em<em&gt; C. Elegans</em&gt; Comportamento Usando um Retenção Ensaio Egg

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Análise da função de células Beta utilizando imagens de cálcio de célula única resolução em ilhotas de Zebrafish

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Um modelo de cultura celular adipócito para estudar o impacto da modulação de proteína e micro-RNA na função adipócito

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Análise da Função Ligase E3 Ubiquitin

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...