Ce travail est soutenu par des subventions de l&#39;Australian National de Santé et Medical Research Council (NHMRC # 559007) et la Juvenile Diabetes Research Foundation (JDRF 4-2006-1025) et le Programme d&#39;infrastructure opérationnelle du gouvernement de Victoria. Nous remercions les membres du Tom Mandel Islet programme de l&#39;équipe de transplantation d&#39;isolement des îlots de fournir les tissus humains. Tissus humains ont été recueillis et utilisés avec l&#39;approbation éthique local (Hôpital Saint-Vincent-de-HREC A 011/04 et de santé St. Vincent HREC-A 135/08).

1. Préparation de tissu de la rate <ol><li> Remarque-tout le matériel humain doit être traité comme potentiellement infectieux et toutes les procédures doivent être menées dans un cabinet à flux laminaire de classe II. Avec des ciseaux et des pinces stériles, retirer les tissus adipeux et fibreux issus de sections rate (~ 1-2 cm) et coupez autant de matériel que possible capsule externe. </li><li> Coupez un petit morceau (1-2 cm 3) de tissu de la rate et placer chaque morceau dans un tube stérile Falcon de 50 ml. </li><li> Snap-congeler les morceaux de tissu par immersion dans l&#39;azote liquide 2. </li><li> Conserver à -80 ° C. Un protocole analogue est adapté à un autre tissu (s). </li></ol> 2. Préparation Islet humaines pour le stockage <ol><li> Îlots de culture dans les médias cmLC. Recueillir des îlots dans un tube de 10 ml à fond conique et laver deux fois dans du PBS par centrifugation à 1500 rpm pendant 5 min. Verser le PBS et vider le tampon résiduel en plaçant le tube inversé brièvement sur une serviette en papier. Veillez à ne paspour déloger les îlots. </li><li> Une fois égoutté, replacez le capuchon du tube et snap-gel dans de l&#39;azote liquide et conserver à -80 ° C. </li></ol> 3. Préparation Extrait <ol><li> Préparer BAW mélange (10:30:60% v / v) et conserver à 4 ° C. </li><li> Retirer le tube de -80 ° C. Décongeler à température ambiante. </li><li> Ajouter suffisamment glacée BAW à couvrir la pièce de tissu. Pour utiliser îlots 3-5 ml. Pour tissu de la rate utiliser 10-20 ml en fonction de la taille de la pièce de tissu. </li><li> Assemblez l&#39;homogénéisateur tissulaire. Nettoyer par «homogénéisation» 10-20 ml d&#39;éthanol à 70% / eau. </li><li> Homogénéiser le tissu en salves multiples, après avoir placé la sonde dans le tube homogénéisateur avec le tissu et la solution BAW. Garder le tube dans un seau à glace par la suite. </li><li> Nettoyer soigneusement l&#39;homogénéisateur entre les échantillons, en homogénéisant 10-20 ml d&#39;éthanol à 70% / eau, puis tampon BAW. Cela permet d&#39;éviter la contamination croisée du tissu entre samples.Dismantleand propre avec 70% d&#39;éthanol / eau après nouse. </li><li> Si un extrait contenant uniquement des matières solubles est nécessaire, centrifuger l&#39;extrait de tissu homogénéisé à 4.000 tours par minute à température ambiante pendant 10 min. Si un extrait brut est plus nécessaire, tourner à 1000 tours par minute à température ambiante pendant 5 min. La technique la plus appropriée pour l&#39;extraction des protéines insolubles dans l&#39;BAW dépend de l&#39;analyse des protéines en aval. Pour une utilisation dans des dosages immunologiques fonctionnels nous vous suggérons de tenter de dissoudre la fraction insoluble dans l&#39;urée 8 M comme nous l&#39;avons déjà constaté que cela soit bien toléré 6. </li><li> Transférer le surnageant dans un tube propre et le mettre sur la glace. </li><li> Déterminer la concentration de protéines dans l&#39;extrait en utilisant un dosage BCA ou similaire. </li></ol> 4. Congelez Extraits de séchage <ol><li> En fonction de la masse de la protéine souhaitée (par exemple, 100 mg par tube), diluer l&#39;homogénat en conséquence et distribuer des aliquotes dans étiqueté stérile 5,0 ml, Falcon (12x75 mm) des tubes. Nous utilisons fréquemment 100 pg / tube. </li><li> Utilisez un 18-20 aiguille de seringue stérile de calibre pour faire 3 trous dans le couvercle de chaque tube. </li><li> Geler les tubes soit en les plaçant sur de la glace sèche pendant environ 10 min ou dans un congélateur à -80 ° C pendant&gt; 1 heure. Conserver à -80 ° C jusqu&#39;au moment de mettre sur le lyophilisateur. </li><li> Allumez le lyophilisateur et ramener la température (-100 ° C). Cela prend environ 30 minutes. </li><li> Selon le volume, la lyophilisation peut être accompli dans les 3 heures, mais nous avons l&#39;habitude laisser nos échantillons durant la nuit. </li><li> Après la fin du cycle de séchage, éteindre la pompe à vide et permettre lentement la pression dans la chambre. Enlever la grille. </li><li> Dans une hotte stérile, enlever les bouchons des tubes perforés et les remplacer par des piles neuves (Falcon 352 032). </li><li> Conserver les tubes à -20 ° C. Les échantillons peuvent être reconstitués dans des milieux de culture, ou des tampons d&#39;autres, et utilisés en fonction ou des essais biochimiques. </li></ol> 5. Les résultats représentatifs La figure 1 montre la coloration d&#39;une protéinegel chargé avec de l&#39;extrait de la rate et du tissu des îlots pancréatiques appauvri (étiqueté acineuse) et purifié îlots humains (îlots étiquetés). Les résultats montrent une bonne représentation de protéines de poids moléculaire différent pour chaque tissu. La capacité de stimuler les extraits de tissus humains prolifération des cellules T a été testée en utilisant un test de prolifération CFSE base 7 (figure 2). Les PBMC utilisé dans cet essai ont été isolés à partir d&#39;une personne avec diabète de type 1. L&#39;ampleur de la réaction est exprimée comme un rapport entre le nombre de cellules CFSE sombres pour 5000 cellules CD4 + CFSE, lumineux sans antigène: des cellules CFSE sombres pour 5000 CD4 +, des cellules CFSE lumineuses avec un antigène provenant d&#39;échantillons en triple 7. Les résultats montrent une faible, mais détectable, en réponse à la prolifération des cellules acineuses (CD1 = 3,5) et une plus forte réponse à l&#39;extrait îlot (6,8). Virus de la grippe inactivé (CDI = 142,6) est inclus en tant quecontrôle positif. <img alt="Figure 1" src="/files/ftp_upload/4230/4230fig1.jpg" /> Figure 1. Gel de protéines. <img alt="Figure 2" src="/files/ftp_upload/4230/4230fig2.jpg" /> Résultats Figure 2. D&#39;un test de prolifération CFSE à base de contre acineuses et extrait îlot. <a href="http://www.jove.com/files/ftp_upload/4230/4230fig2large.jpg" target="_blank">Cliquez ici pour agrandir la figure</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>

Aucun conflit d&#39;intérêt déclaré.

Ce protocole a été développé parce que nous voulions produire un extrait de tissu humain qui est exempt de produits chimiques toxiques tels que les détergents. Plus précisément, nous avons utilisé pour préparer des extraits de tissus humains qui peuvent être utilisés dans des tests de la fonction immunitaire humain in vitro. Les extraits préparés en utilisant ce protocole peut également être reconstitué dans une solution tampon et utilisé pour de nombreuses analyses biochimiques, telle...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> 5 ml 12 x 75 mm tubes en polystyrène stériles </td><td> BD Falcon </td><td> 352054 </td><td></td></tr><tr><td> Bouchons pour tubes tubes en polystyrène (ci-dessus) </td><td> BD Falcon </td><td> 352032 </td><td></td></tr><tr><td> 50ml tubes stériles </td><td> Becton Dickinson </td><td> 352070 </td><td></td></tr><tr><td> Acétonitrile </td><td> Produits chimiques 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> Homogénéisateur: PRO200 </td><td> Bio-stratégie </td><td> 01-01200 </td><td> 10 x 115 mm en dents de scie générateur </td></tr><tr><td> Lyophiliseur </td><td></td><td></td><td> Virtis, de laboratoire 4K </td></tr><tr><td> Aiguille stérile de calibre 18-20 </td><td> Becton Dickinson </td><td> REF 302032 </td><td></td></tr><tr><td> CMRL-1066 Medium </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> Tableau 1. Réactifs et équipements spécifiques.

extraction of tissue antigens for functional assays

La plupart des antigènes cibles de la réponse immunitaire adaptative, reconnus par les cellules B et T, n&#39;ont pas été définis 1. Cela est particulièrement vrai dans les maladies auto-immunes et le cancer 2. Notre objectif est d&#39;étudier les antigènes reconnus par les lymphocytes T humains dans le type 1, diabète maladie auto-immune 1,3,4,5. Pour analyser des cellules T humaines réponses contre les tissus où les antigènes reconnus par les lymphocytes T ne sont pas identifiés, nous avons développé une méthode pour extraire les antigènes protéiques de tissus humains dans un format qui est compatible avec les analyses fonctionnelles 6. Réponses précédemment, des cellules T à des extraits de tissus non purifiés n&#39;a pas pu être mesurée, car les méthodes d&#39;extraction donner un lysat contenant des détergents qui étaient toxiques pour les cellules mononucléées du sang périphérique. Ici, nous décrivons un protocole d&#39;extraction de protéines à partir de tissus humains dans un format qui n&#39;est pas toxique pour les cellules T humaines. Le tissu est homogénéisé dans un mélange de butan-1-ol, l&#39;acétonitrile et water (BAW). La concentration en protéines dans l&#39;extrait de tissu est mesurée et une masse connue de protéine est mise dans des tubes. Après extraction, les solvants organiques sont éliminés par lyophilisation. Extraits de tissus lyophilisés peuvent être conservés jusqu&#39;à leur utilisation. Pour une utilisation dans des tests de la fonction immunitaire, une suspension de cellules du système immunitaire, dans les milieux de culture appropriés, peuvent être ajoutés directement à l&#39;extrait lyophilisé. La production de cytokines et la prolifération des PBMC par, en réponse à des extraits préparés en utilisant cette méthode, ont été faciles à mesurer. Par conséquent, notre méthode permet la préparation rapide des lysats de tissus humains qui peuvent être utilisés comme source d&#39;antigènes dans l&#39;analyse des réponses des cellules T. Nous suggérons que cette méthode va faciliter l&#39;analyse des réponses immunitaires adaptatives des tissus de greffe, le cancer et l&#39;auto-immunité.

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

Extraction of Tissue Antigens for Functional Assays

Un protocole simple de préparation d&#39;extraits de tissus humains pour être utilisées comme source d&#39;antigènes à des cellules T fonctionnelles des essais est décrit. Cette méthode permet réponses des cellules T à des antigènes dérivés de tissus à mesurer<em&gt; In vitro</em&gt;.

Extraction des antigènes tissulaires de tests fonctionnels

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

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10.8K vues.  St. Vincent's Institute of Medical Research. Un protocole simple de préparation d'extraits de tissus humains pour être utilisées comme source d'antigènes à des cellules T fonctionnelles des essais est décrit. Cette méthode permet réponses des cellules T à des antigènes dérivés de tissus à mesurer In vitro.

Vidéo: 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.

Test de transition clair / foncé pour souris

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, ...

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Biologie

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.

Co-actine sédimentation Assay; pour l&#39;analyse des Binding Protein à F-actine

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.

Cristallisant protéines membranaires pour déterminer la structure à l&#39;aide mésophases lipidique

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.

Pharmacologiques et des essais de génétique pour manipuler la régénération de l&#39;Planaires Japonica Dugesia</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.

Utilisation d&#39;un robot pour haut-débit cristallisation des protéines membranaires dans des mésophases lipidiques

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.

Les petits extraits nucléaires pour les dosages fonctionnels de l&#39;expression génétique 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.

Les analyses de viabilité de cellules en culture

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.

Isolement et analyse fonctionnelle des mitochondries de cellules cultivées et de tissus de souris

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.

Préparation de Cores de tissus enrobés de paraffine fixés au formol pour les ARN et d&#39;extraction d&#39;ADN

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.

Étude des Interactions protéine-protéine dans la recherche de l’autophagie

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