Este trabalho é apoiado por concessões do Australian Nacional de Saúde e Pesquisa Médica do Conselho (NHMRC # 559007) eo Juvenile Diabetes Research Foundation (JDRF 4-2006-1025) e do Sistema de Infra-estrutura Operacional do Governo de Victoria. Agradecemos aos membros da Islet Mandel Tom Programa de Transplante Equipe Isolamento Islet para a prestação dos tecidos humanos. Tecidos humanos foram coletadas e utilizadas com aprovação ética local (Hospital São Vicente HREC-A 011/04 e de Saúde de São Vicente HREC-A 135/08).

1. Preparando tecido do baço <ol><li> Nota-todo o material humano devem ser tratados como potencialmente infecciosos e todos os procedimentos devem ser realizados em uma Capela Fluxo Laminar Classe II. Usando tesouras e pinças esterilizadas, remover gordura e tecido fibroso de seções do baço (~ 1-2 cm de tamanho) e retire o máximo de material da cápsula externa possível. </li><li> Corte um pedaço pequeno (1-2 cm 3) de tecido do baço e colocar cada peça em um tubo de 50 ml estéril Falcon. </li><li> Snap-congelar os pedaços de tecido por imersão em N2 líquido. </li><li> Armazenar a -80 ° C. Um protocolo similar é adequado para qualquer outro tecido (s). </li></ol> 2. Preparando Islet Humanos para Armazenamento <ol><li> Ilhotas Cultura na mídia CMRL. Colete ilhotas num tubo de 10 ml de fundo cónico e lavar duas vezes em PBS por centrifugação a 1500 rpm durante 5 min. Decantar a PBS e drenar o tampão residual colocando o tubo invertido brevemente sobre uma toalha de papel. Tenha cuidado para nãopara desalojar as ilhotas. </li><li> Uma vez drenada, re-cap o tubo e encaixe-congelamento em nitrogênio líquido e armazenar a -80 ° C. </li></ol> 3. Preparando Extrato <ol><li> Prepare BAW mix (10:30:60% v / v) e armazenar a 4 ° C. </li><li> Remover o tubo de -80 ° C. Descongelar à temperatura ambiente. </li><li> Adicionar BAW gelado suficiente para cobrir a peça de tecido. Para usar ilhotas 3-5 ml. Para o tecido do baço usar 10-20 ml, dependendo do tamanho do pedaço de tecido. </li><li> Montar o homogeneizador de tecidos. Limpar por &quot;homogeneização&quot; 10-20 ml de etanol a 70% / água. </li><li> Homogeneizar o tecido em explosões múltiplas, após a colocação da sonda para o tubo homogeneizador com o tecido e solução BAW. Manter o tubo em um balde de gelo depois. </li><li> Limpar completamente o homogeneizador entre amostras, por homogeneização de 10-20 ml de etanol a 70% / água e depois de tampão BAW. Isto impede a contaminação cruzada de tecido entre samples.Dismantleand limpa com etanol a 70% / água depois de nóse. </li><li> Se um extracto contendo apenas material solúvel é necessário, centrifugar extracto de tecido homogeneizado a 4000 rpm à temperatura ambiente durante 10 min. Se um extracto bruto é mais necessário, girar a 1000 rpm à temperatura ambiente durante 5 min. A técnica mais apropriada para extrair as proteínas insolúveis em BAW depende da análise a jusante das proteínas. Para utilização em ensaios imunológicos funcionais sugerimos tentar dissolver a fracção insolúvel em ureia 8 M, como já anteriormente foi encontrado que este seja bem tolerada 6. </li><li> Transferir o sobrenadante para um tubo limpo e colocá-lo em gelo. </li><li> Determinar a concentração de proteína no extracto utilizando um ensaio BCA ou similar. </li></ol> 4. Congelar Extractos de secagem <ol><li> Dependendo da massa da proteína desejada (isto é, 100 mg por tubo), dilui-se o homogenato em conformidade e dispensar em alíquotas rotulado 5,0 ml estéril, Falcon (12x75 mm) de tubos. Nós freqüentemente usar 100 mg / tubo. </li><li> Use um 18-20 calibre da agulha de seringa estéril para produzir 3 furos na tampa de cada tubo. </li><li> Congelar tubos ou colocando-os em gelo seco durante ~ 10 min ou no congelador a -80 ° C durante&gt; 1 hora. Loja a -80 ° C até que esteja pronto para colocar no liofilizador. </li><li> Ligue liofilizador e deixar equilibrar (-100 ° C). Isto demora cerca de 30 min. </li><li> Dependendo do volume, a liofilização pode ser concluída dentro de 3 horas, mas nós rotineiramente deixar nossas amostras durante a noite. </li><li> Após a conclusão do ciclo de secagem, desligar a bomba de vácuo e, lentamente, permitir que a pressão dentro da câmara. Remover rack. </li><li> Em um capuz estéril, remover as tampas dos tubos perfurados e substituir por novos (Falcon 352,032). </li><li> Tubos de armazenar a -20 ° C. As amostras podem ser reconstituídos num meio de cultura, ou outros tampões, e usado em ensaios bioquímicos ou função. </li></ol> 5. Resultados representativos A Figura 1 mostra a coloração de uma proteínagel carregado com extracto de tecido do baço e ilhéus pancreáticos empobrecido (rotulado acinar) e purificado ilhéus humanos (ilhotas marcadas). Os resultados mostram uma boa representação de proteínas de peso molecular diferente para cada tecido. A capacidade dos extractos de tecidos para estimular a proliferação de células T humanas foi testado utilizando um ensaio de proliferação CFSE baseado 7 (Figura 2). O PBMC utilizado neste ensaio foram isoladas de um indivíduo com diabetes tipo 1. A magnitude da resposta é expressa como a razão entre o número de células CFSE dim por 5000 células CD4 +, brilhantes, sem antigénio CFSE: de células CFSE dim 5000 por células T CD4 +, células CFSE luminosos com antigénio a partir de amostras triplicadas 7. Os resultados mostram uma fraca, mas a proliferação, detectável em resposta à acinar (CD1 = 3,5) e uma resposta mais forte ao ilhota extracto (6.8). Vírus influenza inactivado (CDI = 142,6) é incluído como umcontrolo positivo. <img alt="Figura 1" src="/files/ftp_upload/4230/4230fig1.jpg" /> Figura 1. Gel Protein. <img alt="Figura 2" src="/files/ftp_upload/4230/4230fig2.jpg" /> Figura 2. Resultados de um ensaio de proliferação CFSE baseado contra acinar e extrato de ilhotas. <a href="http://www.jove.com/files/ftp_upload/4230/4230fig2large.jpg" target="_blank">Clique aqui para ver maior figura</a> .

<ol>
	<li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+to+measuring+human+islet-antigen+specific+T+cell+function+in+type+1+diabetes.">Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes.</a> Clin. Exp. Immunol. 162, 197-209 (2010).</li><li>Beckhove, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+T+cell-based+identification+of+human+tumor+tissue+antigens+by+automated+two-dimensional+protein+fractionation.">Rapid T cell-based identification of human tumor tissue antigens by automated two-dimensional protein fractionation.</a> J. Clin. Invest. 120, 2230-2242 (2010).</li><li>Mannering, S. I., Brodnicki, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+CD4++T-cell+specificity+and+function+in+Type+1+diabetes.">Recent insights into CD4+ T-cell specificity and function in Type 1 diabetes.</a> Expert Review of Clinical Immunology. 3, 557-564 (2007).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+A-chain+of+insulin+is+a+hot-spot+for+CD4++T+cell+epitopes+in+human+type+1+diabetes.">The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.</a> Clin. Exp. Immunol. 156, 226-231 (2009).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insulin+A-chain+epitope+recognized+by+human+T+cells+is+posttranslationally+modified.">The insulin A-chain epitope recognized by human T cells is posttranslationally modified.</a> J. Exp. Med. 202, 1191-1197 (2005).</li><li>Moon, H. C., Joffe, M., Thomas, H. E., Kay, T. W. H., Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+extracting+tissue+proteins+for+use+in+lymphocyte+function+assays.">A method for extracting tissue proteins for use in lymphocyte function assays.</a> Journal of Immunological Methods. 359, 56-60 (2010).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J. Immunol. Methods. 283, 173-183 (2003).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+Cell+Proliferation+in+Response+to+GAD+and+Proinsulin+in+Healthy,+Pre-diabetic,+and+Diabetic+Donors.">CD4+ T Cell Proliferation in Response to GAD and Proinsulin in Healthy, Pre-diabetic, and Diabetic Donors.</a> Ann. N.Y. Acad. Sci. 1037, 16-21 (2004).</li><li>Mannering, S. I., Purcell, A. W., Honeyman, M. C., McCluskey, J., Harrison, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T-cells+recognise+N-terminally+Fmoc-modified+peptide.">Human T-cells recognise N-terminally Fmoc-modified peptide.</a> Vaccine. 21, 3638-3646 (2003).</li><li>Peakman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+preparations+of+GAD65,+proinsulin,+and+the+islet+tyrosine+phosphatase+IA-2+for+use+in+detection+of+autoreactive+T-cells+in+type+1+diabetes:+report+of+phase+II+of+the+Second+International+Immunology+of+Diabetes+Society+Workshop+for+Standardization+of+T-cell+assays+in+type+1+diabetes.">Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the Second International Immunology of Diabetes Society Workshop for Standardization of T-cell assays in type 1 diabetes.</a> Diabetes. 50, 1749-1754 (2001).</li><li>Honey, K., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+cysteine+proteases+regulate+antigen+presentation.">Lysosomal cysteine proteases regulate antigen presentation.</a> Nat. Rev. Immunol. 3, 472-482 (2003).</li></ol>

Não há conflitos de interesse declarados.

Este protocolo foi desenvolvido porque queríamos gerar um extrato de tecido humano que estava livre de produtos químicos tóxicos como detergentes. Especificamente utilizou-se para preparar os extractos de tecido humano que podem ser utilizados em ensaios de função imunitária humana, in vitro. Extractos preparados utilizando este protocolo pode igualmente ser reconstituída em qualquer tampão e usados ​​para muitas análises bioquímicas, tais como cromatografia de Western blotting ou líquid...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> 5 ml 12 x 75 mm tubos de polistireno estéreis </td><td> BD Falcon </td><td> 352054 </td><td></td></tr><tr><td> Tampas para tubos de tubos de poliestireno (acima) </td><td> BD Falcon </td><td> 352032 </td><td></td></tr><tr><td> 50ml tubos estéreis </td><td> Becton Dickinson </td><td> 352070 </td><td></td></tr><tr><td> Acetonitrila </td><td> Chemicals Mallinckradt </td><td> 2856-10 </td><td></td></tr><tr><td> Butan-1-ol </td><td> Sigma Aldrich </td><td> 537993-IL </td><td></td></tr><tr><td> Homogeneizador: PRO200 </td><td> Bio-estratégia </td><td> 01-01200 </td><td> 10 x 115 mm gerador de dente de serra </td></tr><tr><td> Liofilizador </td><td></td><td></td><td> Virtis, bancada 4K </td></tr><tr><td> Agulha estéril calibre 18-20 </td><td> Becton Dickinson </td><td> REF 302032 </td><td></td></tr><tr><td> CMRL-1066 Meio </td><td> Sigma </td><td> C0422 </td><td></td></tr><tr><td> PBS </td><td> Sigma </td><td> D8537 </td><td></td></tr></tbody></table> Tabela 1. Reagentes específicos e de equipamento.

extraction of tissue antigens for functional assays

Muitos dos alvos de antigénio de resposta imune adaptativa, reconhecidos por células T e B, não foram definidas 1. Isto é particularmente verdadeiro em doenças auto-imunes e cancro 2. O nosso objectivo é o de investigar os antigénios reconhecidos pelas células T humanas do tipo de doença auto-imune uma diabetes 1,3,4,5. Para a análise de células T humanas respostas contra o tecido, onde os antigénios reconhecidos pelas células T são identificadas não foi desenvolvido um método para extrair os antigénios de proteína a partir de tecido humano, em um formato que é compatível com ensaios funcionais 6. Respostas previamente, de células T para extractos de tecido não purificados não puderam ser medidos por métodos de extracção de produzir um lisado que continha detergentes que foram tóxicas para as células mononucleares do sangue periférico. Aqui descrevemos um protocolo para a extracção de proteínas a partir de tecidos humanos, em um formato que não é tóxico para as células T humanas. O tecido é homogeneizado numa mistura de butan-1-ol, acetonitrilo e water (BAW). A concentração de proteína no extracto de tecido é medida e uma massa conhecida de proteína é separado em alíquotas para tubos. Depois da extracção, os solventes orgânicos são removidos por meio de liofilização. Extractos de tecidos liofilizados podem ser armazenados até serem necessários. Para utilização em ensaios da função imune, uma suspensão de células do sistema imunológico, em meios de cultura apropriados, podem ser adicionados directamente ao extracto liofilizado. A produção de citocinas e a proliferação de PBMC em resposta a extractos preparados utilizando este método, foram facilmente medido. Por isso, o nosso método permite a preparação rápida de lisados ​​de tecido humano que podem ser utilizados como uma fonte de antigénios na análise de respostas de células T. Sugerimos que este método irá facilitar a análise das respostas imunes adaptativas para os tecidos em transplantes, cancro e auto-imunidade.

Watch this Scientific Journal Video about Extraction of Tissue Antigens for Functional Assays at JoVE.com

Extraction of Tissue Antigens for Functional Assays

Um protocolo simples para a preparação de extractos de tecido humano para ser usado como uma fonte de antigénios de células T funcionais de ensaios está descrito. Este método permite a respostas das células T aos antigénios derivados de tecido a ser medido<em&gt; In vitro</em&gt;.

Extracção do Tissue Antigens para Ensaios Funcionais

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

extraction-of-tissue-antigens-for-functional-assays

Research

JoVE Journal

Biology

10.8K visualizações.  St. Vincent's Institute of Medical Research. Um protocolo simples para a preparação de extractos de tecido humano para ser usado como uma fonte de antigénios de células T funcionais de ensaios está descrito. Este método permite a respostas das células T aos antigénios derivados de tecido a ser medido In vitro.

Vídeo: Extraction of Tissue Antigens for Functional Assays

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Claro / escuro Teste de transição para Ratos

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Biologia

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Ensaio Co Sedimentação; para a Análise da proteína de ligação a F-Actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Cristalizando Proteínas de Membrana para determinação da estrutura usando mesofases lipídico

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Farmacológica e funcional Ensaios genética para manipular Regeneração da planária Dugesia japonica</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

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 ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

Os pequenos extratos nucleares para ensaios funcionais da expressão gênica Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Ensaios de viabilidade para células em cultura

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolamento e Análise Funcional de mitocôndrias de células cultivadas e mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Preparação de Núcleos de tecido parafina-encaixados formalina-fixados para RNA e DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Estudo das interações proteína-proteína em pesquisa de autofagia

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...