Gostaríamos de agradecer ao Dr. S. Ressler (Departamento de Biologia Molecular e Celular, Baylor College of Medicine) para sugerir o uso de proteína concentradores para aumentar a concentração de MMPs em sobrenadante de cultura de células. Este projeto foi suportado por concessões do Elder Clifford Sra. White Graham Fundo de Investigação Dotado eo NIAMS NIH / (AR059838) para CB. O conteúdo é da exclusiva responsabilidade dos autores e não representam, necessariamente, a posição oficial do NIH.

1. Carregando e executando o Gel <ol><li> Todas as amostras devem ser preparadas adequadamente para manter a função das enzimas e utilizado imediatamente após a coleta ou armazenado congelado a -80 ° C. As amostras não devem conter agentes redutores (como uma β-mercaptoetanol) ou ser fervida antes de carregá-gel. </li><li> Abra a bolsa que contém um gel dentro de uma gaveta, lavar a cassete com água deionizada. <ul><li> Remover a fita protetora da parte inferior da cassete eo pente de cima do gel. </li><li> Lave os poços três vezes com tampão de corrida (diluído em água deionizada 1X). </li><li> Coloque o gel para o Mini-Cell, garantindo que o menor lado da cassete faces para dentro. Encaixado com o Wedge Tension Gel. O Mini-Cell permite executar um ou dois géis em paralelo. </li><li> Encha a câmara (no interior) superior com 1X tampão de corrida acima do nível do poço, verificar se há vazamentos. Em caso de vazamentos, retire o tampão e reposicionar o gel. </li><li> Encha a câmara baixa com 1X tampão de corrida. </li></ul></li><li> Carga de 10 L de marcador molecular de proteínas em um poço. <ul><li> Misture uma quantidade igual de gel de carregamento de buffer e de amostra e de carga nos poços do gel utilizando gel de carregamento de dicas (alterada entre cada amostra). Estes poços podem ser carregados com até 20 total mL. </li></ul></li><li> Coloque a tampa sobre a Célula Mini-e conecte os cabos eletrodo para o fornecimento de energia. Mudar a fonte de alimentação e configurá-lo para funcionar em 125 V constante por 90 minutos. Verificar a formação de pequenas bolhas no fio da câmara baixa, indicando a circulação atual. </li><li> Ao executar o seu primeiro gel, monitorar o progresso da migração a cada 15 minutos, utilizando o azul de bromofenol incluído no tampão de carregamento como um indicador. Deixe a executar até que o gel corante indicador atinge o fundo do gel. </li></ol> 2. Renaturing e Desenvolvimento do Gel <ol><li> Para cada gel, preparar 100 ml de 1X tampão renaturing e 200 mL de tampão de desnaturação, tanto em água deionizada. </li><li> Quando o corante azul de bromofenol rastreamento atinge o fundo do gel, mude a fonte de alimentação desligada, abrir o celular Mini-, e remover o gel. Separar os dois lados da cassete usando a faca gel (ou uma espátula de pesagem). Cortar um canto para marcar a direção do gel. </li><li> Remova cuidadosamente o gel da gaveta e coloque em um recipiente com 100 mL de tampão renaturing. Incubar por 30 minutos em temperatura ambiente com agitação suave. </li><li> Remova o tampão renaturing e adicionar 100 mL de buffer para o desenvolvimento de gel. Incubar por 30 minutos em temperatura ambiente com agitação suave. </li><li> Remova o tampão desenvolvimento e adicionar mais 100 mL de tampão para o desenvolvimento de gel. Incubar overnight (16-18 horas) a 37 ° C. </li><li> Remova o tampão desenvolvimento e lavar três vezes (5 minutos cada) com água deionizada sob agitação suave na temperatura ambiente. </li><li> Digitalizar o gel para salvar o posicionamento exato das bandas padrão de proteínas como eles vão se tornar menos visíveis ou não após coloração do gel. </li><li> Manchar o gel pela adição de 20 mL de SimplyBlue Safestain ao gel. Incubar durante uma hora à temperatura ambiente sob agitação suave. </li><li> Remover o SafeStain SimplyBlue e de mancha o gel em 100 mL ou água deionizada por mais uma hora em temperatura ambiente sob agitação suave. </li><li> Para melhores resultados, substitua com água deionizada fresco e incubar por mais uma hora ou mais à temperatura ambiente sob agitação suave. </li></ol> 3. Análise de Dados <ol><li> Remova cuidadosamente o gel da água e coloque em um protetor de plástico. </li><li> Digitalizar o gel com uma resolução de 300 dpi ou superior. Salve a imagem no formato TIFF (Figura 1A). </li><li> Medir intensidades banda com ImageJ (ou outro software similar). <ul><li> Abra o arquivo TIFF no ImageJ (Figura 1A). </li><li> Visualize em preto e branco, selecionando &quot;Image&gt;&gt; Tipo de 8 bits&quot; (Figura 1B). </li><li> Use a ferramenta de seleção retangular para delinear a primeira banda, desenhando um retângulo, pelo menos, duas vezes maior do que larga (este é um requisito de software). </li><li> Pressione &quot;1&quot; ou selecione &quot;Analisar&gt; Géis&gt; lane Selecione primeiro&quot; ea banda serão delineadas. </li><li> Um novo retângulo aparecerá, movê-lo para a banda e escolha a opção &quot;Analisar&gt; Géis&gt; lane Select next&quot;. Repita até que todas as bandas são selecionados (Figura 1B). </li><li> Pressione &quot;3&quot; ou selecione &quot;Analisar&gt; Géis pistas&gt; Plot&quot; para gerar o mapa do perfil de cada banda (Figura 1C). </li><li> Use a seleção de linha reta (já deve estar selecionado automaticamente) para desenhar linhas de base para que o pico de interesse é uma área completamente fechado. </li><li> Selecione a ferramenta varinha e clique dentro de cada pico para selecioná-lo. </li><li> Selecione &quot;Analisar&gt; Géis picos&gt; Label&quot; para obter uma tabela com a área de cada pico selecionado. Estes dados podem ser plotados como tal (F1D IGURA) ou pode ser normalizada para o valor de uma banda escolhida. Esta normalização é especialmente importante quando o agrupamento de vários valores géis replicar para determinar a significância estatística dos resultados. </li></ul></li></ol> 4. Resultados representante Mostramos um gel com 11 poços carregado com sobrenadantes de células diferentes de cultura contendo diferentes quantidades de MMP-2 (Figura 1A). Observação direta deste gel mostra diferenças óbvias em MMP-2 concentrações entre alguns dos poços. Por exemplo, fica claro que os poços # 4 e 5 contêm muito mais MMP-2 do que bem # 11 ou até mesmo bem # 10. Para a quantificação objetivo de bandas que têm usado com o software de densitometria ImageJ (Figura 1B-D), confirmando uma diferença de cerca de 4 vezes na MMP-2 montantes entre bem # 11 e # poços 4 e 5 e uma diferença de aproximadamente 2 vezes na MMP-2 montantes entre bem # 10 e # poços 4 e 5. <img src="/files/ftp_upload/2445/2445fig1.jpg" alt="Figura 1" /> Figura 1. Detecção de MMP-2 por zimografia gelatina. Uma imagem, digitalizada de um gel de gelatina. Números são bem indicado na parte superior do gel. B gel, mesmo que em um mostradas em preto e branco para a densitometria. Cada banda foi selecionada com um retângulo no ImageJ. C, dois exemplos de perfis de densitometria para o gel mostrado em A e B (bandas # 4 e # 11). Uma linha reta foi traçada na base de cada pico para criar uma área fechada. D, Lote das áreas de pico para o gel mostrado em A e B antes (esquerda) e após a normalização (direita) para a densidade da banda # 1. <img src="/files/ftp_upload/2445/2445fig2.jpg" alt="Figura 2" /> Figura 2. Esta figura demonstra como zimografia pode ser usado como uma técnica semi-quantitativa. Temos carregado diluições em série (passos de 1:1 diluições) em 7 poços consecutivos e mediram a densidade de banda para os dois pró-MMP-2 e MMP-2.

<ol>
	<li>Luo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+matrix+metalloproteinases+in+the+morphogenesis+of+the+cerebellar+cortex.">The role of matrix metalloproteinases in the morphogenesis of the cerebellar cortex.</a> Cerebellum. 4, 239-245 (2005).</li><li>Pasternak, B., Aspenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloproteinases+and+their+inhibitors+-+diagnostic+and+therapeutic+opportunities+in+orthopedics.">Metalloproteinases and their inhibitors - diagnostic and therapeutic opportunities in orthopedics.</a> Acta Orthop. 80, 693-703 (2009).</li><li>Kessenbrock, K., Plaks, V., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases:+regulators+of+the+tumor+microenvironment.">Matrix metalloproteinases: regulators of the tumor microenvironment.</a> Cell. 141, 52-67 (2010).</li><li>Lagente, V., Boichot, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+matrix+metallopreoteinases+in+the+inflammatory+process+of+respiratory+diseases.">Role of matrix metallopreoteinases in the inflammatory process of respiratory diseases.</a> J. Mol. Cell. Cardiol. 48, 440-444 (2010).</li><li>Rodr&#237;guez, D., Morrison, C. J., Overall, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metallopreoteinases:+what+do+they+not+do?+New+substrates+and+biological+roles+identified+by+murine+models+and+proteomics.">Matrix metallopreoteinases: what do they not do? New substrates and biological roles identified by murine models and proteomics.</a> Biochim. Biophys. Acta. 1803, 39-54 (2010).</li><li>Bourboulia, D., Stetler-Stevenson, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases+(MMPs)+and+tissue+inhibitors+of+metalloproteinases+(TIMPs):+positive+and+negative+regulators+in+tumor+cell+adhesion.">Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): positive and negative regulators in tumor cell adhesion.</a> Semin. Cancer Biol. , (2010).</li><li>Heussen, C., Dowdle, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+analysis+of+plasminogen+activators+in+polyacrylamide+gels+containing+sodium+dodecyl+sulfate+and+copolymerized+substrates.">Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates.</a> Analytical Biochem. , 102-196 (1980).</li><li>Lombard, C., Saulnier, J., Wallach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+of+matrix+metalloproteinases+(MMPs)+activities:+a+review.">Assays of matrix metalloproteinases (MMPs) activities: a review.</a> Biochimie. 87, 265-272 (2005).</li><li>Snoek-van Beurden, P. A., Von den Hoff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymographic+techniques+for+the+analysis+of+matrix+metalloproteinases+and+their+inhibitors.">Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors.</a> Biotechniques. 38, 73-83 (2005).</li><li>Kupai, K., Szucs, G., Cseh, S., Hajdu, I., Csonka, C., Csont, T., Ferdinandy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+activity+assays:+importance+of+zymography.">Matrix metalloproteinase activity assays: importance of zymography.</a> J. Pharmacol. Toxicol. Methods. 61, 205-209 (2010).</li><li>Kleiner, D. E., Stetler-Stevenson, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+zymography:+detection+of+pictogram+quantities+of+gelatinases.">Quantitative zymography: detection of pictogram quantities of gelatinases.</a> Anal. Biochem. 218, 325-329 (1994).</li><li>Yu, W. H., Woessner, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin-enhanced+zymographic+detection+of+matrilysin+and+collagenases.">Heparin-enhanced zymographic detection of matrilysin and collagenases.</a> Anal. Biochem. 293, 38-42 (2001).</li></ol>

Não há conflitos de interesse declarados.

Nós demonstramos como realizar análise zymographic de MMP-2 em sobrenadante de cultura de células. A quantidade de amostra para carregar em um gel deve ser determinada empiricamente, dependendo da origem e MMP de interesse. Carregamento muito pouco vai impedir a detecção ao carregar demais pode levar à saturação como há substrato somente tanto uma protease pode digerir na área da banda. Se necessário, a amostra pode ser diluída em água deionizada antes da mistura com tampão de carregamento ou o tempo de desenvolvimento podem reduzido de overnight a 4 horas. Também é possível concentrar as proteínas em uma solução utilizando concentradores (por exemplo, catálogo Millipore # UFC803024). Se bandas permanecem pouco visíveis, pode ser necessário para desenvolver o gel por um longo período de tempo, até um máximo de 48 horas. Ao preparar as amostras de sobrenadantes de cultura de tecido, deve-se notar que o soro fetal bovino (e outros soros) contém MMPs que podem afetar os resultados. Por essa razão, coletamos todos os nossos sobrenadantes após cultura em meio livre de soro. As lavagens descritos nos parágrafos 2.9 e 2.10 do protocolo são necessárias para remover o fundo de coloração de bandas digeridas. Vezes mais lavar irá remover mais fundo, mas também dim a coloração do substrato ao longo do gel. Se lavar mais vezes são necessárias, recomendamos a varredura do gel a cada poucas horas para selecionar o scan com o melhor contraste. Esta técnica pode ser utilizada para detectar outras MMPs. Por exemplo, MMP-9 pode ser detectado em gel de gelatina, e também em menor escala MMP-1, MMP-8 e MMP-13 como a gelatina não é o seu substrato preferencial. MMP-1 e MMP-13 são melhor detectadas em zimografia colágeno, enquanto a caseína é o substrato preferido para a MMP-11 e também permite a detecção de MMP-1, MMP-3, MMP-7, MMP-12 e MMP-13 9. MMP-7 (matrilisina) e colagenases (MMP-1 e MMP-13) são difíceis de detectar quando presente em níveis baixos em caseína ou géis de gelatina. Adição de heparina ao exemplo em tempo de carregamento gel melhora significativamente o limite de detecção para MMP-7. Para MMP-1 e MMP-13, a heparina deve ser adicionado ao gel após a corrida eletroforética já está em andamento 12. Desde zimografia medidas atividade enzimática após desnaturação e renaturação das enzimas, ele irá medir a atividade de todas as MMPs presentes na amostra. Isto inclui as enzimas, pró-enzimas, enzimas e obrigado a inibidores endógenos (por exemplo, TIMP-2). No entanto, é possível determinar o nível de ativação MMP na amostra, comparando a densidade da banda da enzima ativa e do da pró-enzima, que terá um peso molecular um pouco maior. Zimografia muitas vezes não é suficiente para identificar uma MMP. Comparação do nível de migração de uma MMP com conhecidos padrões de peso molecular não ajuda na identificação, mas deve-se notar que algumas dessas normas contêm um agente redutor e que, quando usado sob condições não-redução que pode indicar diferentes pesos moleculares 9. Uma opção é carregar um MMP solúvel recombinante no mesmo gel que as amostras de teste. MMPs pode, contudo, ser associado com outras proteínas, induzindo uma mudança no peso molecular aparente. Inibidores seletivos MMP pode ser adicionado ao gel (ou parte de um corte pela metade gel) durante a incubação em tampão desenvolvimento como ferramentas farmacológicas para a identidade do MMP de interest.A segunda técnica (Western Blot, imunocitoquímica, ou ELISA) é recomendada para ajudar na identificação da MMP de interesse. Note-se que os limites de detecção para Western Blot são frequentemente muito inferiores aos dos géis zymographic, podendo levar a resultados falso-negativos quando se usa esta técnica.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Xcell SureLock Mini-Cell CE mark electrophoresis apparatus</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Power supply (model 302)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>93000-744</td>
<td>&nbsp;</td>
<tr>
<td>Novex 10% gelatin zymogram gels, 1.0 mm, 12 wells</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC61752BOX</td>
<td>&nbsp;</td>
<tr>
<td>Blue Juice gel loading buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10816015</td>
<td>&nbsp;</td>
<tr>
<td>Gel loading tips</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53509-015</td>
<td>&nbsp;</td>
<tr>
<td>Protein molecular weight standard</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC5800</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tris-Glycine-SDS running buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2675</td>
<td>&nbsp;</td>
<tr>
<td>Novex zymogram renaturing buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2670</td>
<td>&nbsp;</td>
<tr>
<td>Novex zymogram developing buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2671</td>
<td>&nbsp;</td>
<tr>
<td>SimplyBlue SafeStain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6060</td>
<td>&nbsp;</td>
<tr>
<td>Epson Perfection 4490 Photo Scanner</td>
<td>&nbsp;</td>
<td>Amazon</td>
<td>n/a</td>
<td>&nbsp;</td>
<tr>
<td>ImageJ software</td>
<td>&nbsp;</td>
<td>http://rsbweb.nih.gov/ij/ </td>
<td>n/a</td>
<td>Authored by W. Rasband, NIH/NIMH</td>
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detection of functional matrix metalloproteinases by zymography

Metaloproteinases de matriz (MMPs) são endopeptidases contendo zinco. Eles degradam proteínas por clivagem de ligações peptídicas. Mais de vinte MMPs foram identificados e são separados em seis grupos com base em sua estrutura e especificidade de substrato (colagenases, gelatinases, tipo de membrana [MT-MMP], estromelisinas, matrilisinas, e outros). MMPs desempenham um papel crítico na invasão celular, a degradação da cartilagem, remodelação do tecido, cicatrização de feridas, e embriogênese. Eles, portanto, participar em ambos os processos normais e na patogênese de muitas doenças, como artrite reumatóide, câncer ou doença pulmonar obstrutiva crônica 1-6. Aqui, vamos nos concentrar em MMP-2 (gelatinase A, colagenase tipo IV), a MMP amplamente expressa. Vamos demonstrar como detectar MMP-2 em sobrenadante de cultura de células por zimografia, uma técnica comumente usada, simples, e ainda assim muito sensível descrita pela primeira vez em 1980 por C. Heussen e Dowdle EB 7-10. Esta técnica é semi-quantitativo, portanto pode ser usada para determinar os níveis de MMP em amostras de teste quando concentrações conhecidas de MMP recombinante são carregados no mesmo gel 11. Soluções contendo MMPs (por exemplo, sobrenadantes de cultura de células, urina ou soro) são carregados em um gel de poliacrilamida contendo dodecil sulfato de sódio (SDS, para linearizar as proteínas) e gelatina (substrato para a MMP-2). O tampão de amostra é projetado para aumentar a viscosidade da amostra (para facilitar o carregamento gel), fornecem um corante de rastreamento (azul de bromofenol, para monitorar a migração da amostra), fornecem moléculas de desnaturação (para linearizar proteínas), e controlar o pH da amostra. Proteínas são, então, permissão para migrar sob uma corrente elétrica em um tampão de corrida projetado para fornecer uma taxa de migração constante. A distância de migração é inversamente correlacionada com o peso molecular da proteína (pequenas proteínas se movem mais rapidamente através do gel de proteínas e, portanto, fazer grandes migrar ainda mais para baixo do gel). Após a migração, o gel é colocado em um buffer renaturing para permitir que as proteínas para recuperar a sua estrutura terciária, necessárias para a atividade enzimática. O gel é então colocada em um buffer de desenvolvimento concebido para permitir a protease para digerir seu substrato. O buffer de desenvolvimento também contém p-aminophenylmercuric acetato (APMA) para ativar a não proteolíticos pró-MMPs em MMPs ativas. O próximo passo consiste em manchar o substrato (gelatina no nosso exemplo). Depois de lavar o excesso de corante fora do gel, as áreas de digestão protease aparecem como faixas claras. Quanto mais clara a banda, mais concentrada a protease que ele contém. Intensidade de coloração da banda pode ser determinada por densitometria, utilizando um software, como ImageJ, permitindo a comparação da amostra.

Watch this Scientific Journal Video about Detection of Functional Matrix Metalloproteinases by Zymography at JoVE.com

Detection of Functional Matrix Metalloproteinases by Zymography

Este protocolo descreve um ensaio baseado em atividades para a detecção de metaloproteinases de matriz em sobrenadante de cultura ou fluidos corporais.

Detecção de Metaloproteinases da Matriz Funcional por zimografia

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

detection-of-functional-matrix-metalloproteinases-by-zymography

Nanyang Technological University

Research

JoVE Journal

Biology

83.6K visualizações.  Baylor College of Medicine. Este protocolo descreve um ensaio baseado em atividades para a detecção de metaloproteinases de matriz em sobrenadante de cultura ou fluidos corporais.

Vídeo: Detection of Functional Matrix Metalloproteinases by Zymography

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Biologia

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

Detecção de modificações pós-translacionais sobre Native Nucleossomos Intact por ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

A utilização de um robô para alta capacidade de cristalização de proteínas de membrana em mesofases lipídicas

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Colheita e Cryo-resfriamento cristais de proteínas de membrana crescida no mesofases lipídicas para a determinação da estrutura de Cristalografia Macromolecular

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Detecção de RNA viral por fluorescência<em&gt; In situ</em&gt; Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Melhorar a taxa de sucesso de cristalização de proteínas pela Random Microseed Matrix Screening

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

Detecção de Vivo<em&gt; Escherichia coli</em&gt; O157: H7 Células por PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Detecção de proteínas de ligação a ARN pela<em&gt; In Vitro</em&gt; RNA Pull-down em Cultura adipócito

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...