Diese Arbeit wird durch Zuschüsse aus dem Australian National Health and Medical Research Council (NHMRC # 559007) und der Juvenile Diabetes Research Foundation (JDRF 4-2006-1025) und der betrieblichen Infrastruktur Schema des viktorianischen Regierung unterstützt. Wir danken den Mitgliedern des Tom Mandel Inseltransplantation Programm Inselisolierung Team für die Bereitstellung der menschlichen Geweben. Menschliche Gewebe wurden gesammelt und mit lokalen ethischen Zulassung verwendet (St. Vincent Hospital HREC-A 011/04 und St. Vincent Gesundheit HREC-A 135/08).

Ein. Vorbereiten Milzgewebe <ol><li> Hinweis-all menschlichem Material sollten als potenziell infektiös und alle Verfahren sollten in einer Klasse II Laminar Flow Cabinet durchgeführt werden behandelt werden. Verwendung steriler Schere und Pinzette Fett und Bindegewebe aus Milz Abschnitte (~ 1-2 cm groß) und schneiden Sie so viel von der äußeren Kapsel Material wie möglich. </li><li> Schneiden Sie ein kleines Stück (1-2 cm 3) von Milzgewebe und platzieren Sie jedes Stück in einem sterilen 50 ml-Falcon-Röhrchen. </li><li> Snap-frieren die Gewebestücke durch Eintauchen in flüssigen N 2. </li><li> Lagerung bei -80 ° C. Ein ähnliches Protokoll ist für andere Gewebe (n). </li></ol> 2. Herstellung von Human Islet für Storage <ol><li> Kultur Inseln in CMRL Medien. Sammle Inselchen in einem 10 ml konischen Boden Rohr und waschen zweimal in PBS durch Zentrifugation bei 1.500 rpm für 5 min. Abgießen, PBS und lassen Sie das restliche Puffer, indem das invertierte Rohr kurz auf einem Papiertuch. Achten Sie darauf,um die Inseln zu vertreiben. </li><li> Nach dem Abtropfen, re-cap die Röhre und Snap-freeze in flüssigem Stickstoff eingefroren und bei -80 ° C. </li></ol> 3. Vorbereiten Extract <ol><li> Bereiten BAW-Mix (10:30:60% v / v) und bei 4 ° C. </li><li> Entfernen Sie den Schlauch von -80 ° C. Bei Raumtemperatur auftauen. </li><li> Ausreichend eiskalten BAW, um das Stück von Gewebe zu decken. Für Inselchen verwenden 3-5 ml. Für Milzgewebe verwenden 10-20 ml je nach Größe des Stückes von Gewebe. </li><li> Montieren Sie den Gewebehomogenisator. Reinigen durch &quot;Homogenisieren&quot; 10-20 ml 70% Ethanol / Wasser. </li><li> Homogenisieren des Gewebes in mehrere Bursts, nach dem Aufsetzen des Homogenisators Sonde in das Rohr mit dem Gewebe und BAW Lösung. Halten Sie das Rohr in einem Eis-Eimer danach. </li><li> Reinigen Sie den Homogenisator zwischen den Proben, durch Homogenisieren 10-20 ml 70% Ethanol / Wasser und dann BAW-Puffer. Dies verhindert Kreuzkontamination von Gewebe zwischen samples.Dismantleand sauber mit 70% Ethanol / Wasser nach unsE. </li><li> Wenn ein Extrakt, der nur lösliche Material benötigt, zentrifugieren homogenisierte Gewebeextrakt bei 4.000 Upm bei RT für 10 min. Wenn ein Rohextrakt mehr erforderlich ist, bei 1000 Upm bei RT für 5 min drehen. Die am besten geeignete Methode zur Extraktion der BAW-unlösliche Proteine ​​hängt vom nachgeschaltete Analyse der Proteine. Für den Einsatz in der funktionellen immunologischen Assays würden wir vorschlagen, versuchen, die unlösliche Fraktion in 8 M Harnstoff aufzulösen, wie wir zuvor gefunden haben dies gut verträglich zu sein 6. </li><li> Überstand in ein sauberes Röhrchen und legte es auf Eis. </li><li> Bestimmung der Konzentration von Protein in der Extrakt unter Verwendung eines BCA-Assay oder ähnliches. </li></ol> 4. Gefriertrocknung Auszüge <ol><li> Je nach der Masse des Proteins erforderlich (dh, 100 ug pro Röhrchen), verdünnt das Homogenat und abzugeben entsprechend Aliquots in 5,0 ml steriler markierten, Falcon (12x75 mm) Tuben. Wir verwenden häufig 100 ug / Rohr. </li><li> Verwenden Sie eine 18-20 Gauge sterilen Injektionsnadel bis 3 Löcher in der Kappe jeder Röhre zu machen. </li><li> Frieren Rohre entweder, indem sie auf Trockeneis für ~ 10 min oder in einem -80 ° C Gefrierschrank für&gt; 1 Stunde. Lagerung bei -80 ° C bis zum auf dem Lyophilisator setzen. </li><li> Schalten Sie Gefriertrockner und ermöglichen äquilibrieren (-100 ° C). Dieser Vorgang dauert ca. 30 min. </li><li> Je nach Volumen kann Gefriertrocknung innerhalb von 3 Stunden abgeschlossen sein, aber wir routinemäßig verlassen unsere Proben über Nacht. </li><li> Nach Beendigung des Trocknungszyklus, Abschaltung Vakuumpumpe und langsam ermöglichen Druck in der Kammer. Entfernen Rack. </li><li> In einer sterilen Haube, entfernen Sie die Lochkappen aus den Rohren und durch neue ersetzen (Falcon 352.032). </li><li> Röhrchen bei -20 ° C. Proben können in Kulturmedien oder andere Puffer rekonstituiert werden, und verwendet in Funktion oder biochemischen Assays. </li></ol> 5. Repräsentative Ergebnisse Abbildung 1 zeigt die Färbung eines ProteinsGel mit Extrakt aus der Milz und Inselchen erschöpft Pankreasgewebe (beschriftet acinar) und gereinigten menschlichen Inseln (mit Inseln) geladen. Die Ergebnisse zeigen eine gute Darstellung der Proteine ​​verschiedener Molekulargewicht für jedes Gewebe. Die Kapazität Gewebeextrakten die menschliche T-Zell-Proliferation zu stimulieren, wurde getestet unter Verwendung eines CFSE basierenden Proliferationsassay 7 (Abbildung 2). Die PBMC in diesem Test verwendet wurden von einem Individuum mit Diabetes Typ 1 isoliert. Das Ausmaß der Reaktion wird als Verhältnis der Anzahl der Zellen pro CFSE dim 5000 CD4 +, CFSE hellen Zellen ohne Antigen exprimiert: der CFSE dim Zellen pro 5000 CD4 +, CFSE hellen Zellen mit Antigen aus Dreifachproben 7. Die Ergebnisse zeigen eine schwache, aber nachweisbare, Proliferation als Reaktion auf Azinuszellen (CD1 = 3,5) und eine stärkere Reaktion auf islet Extrakt (6,8). Inaktivierten Influenza-Virus (CDI = 142,6) als enthaltenpositive Kontrolle. <img alt="Abbildung 1" src="/files/ftp_upload/4230/4230fig1.jpg" /> Abbildung 1. Protein-Gel. <img alt="Abbildung 2" src="/files/ftp_upload/4230/4230fig2.jpg" /> Abbildung 2. Ergebnisse einer CFSE-basierte Proliferationsassay gegen acinar und Inselchen Extrakt. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/4230/4230fig2large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung zu sehen</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>

Keine Interessenskonflikte erklärt.

Dieses Protokoll wurde entwickelt, weil wir einen Auszug aus menschlichem Gewebe, die frei von giftigen Chemikalien wie Wasch war erzeugen wollte. Insbesondere haben wir es verwendet, um Auszüge aus menschlichem Gewebe, die in Tests des menschlichen Immunsystems in vitro verwendet werden vorzubereiten. Extrakte hergestellt unter Verwendung dieses Protokolls können gleichermaßen in einem Puffer rekonstituiert werden und für viele biochemische Analysen, wie Western-Blot oder Flüssigkeitschromatographie. Dies macht diese Technik für viele nachgelagerte Anwendungen. Mit unserem Protokoll die Antwort auf Gewebeextrakten sind nicht stark. Es wird erwartet, weil wir für responses to &#39;self&#39; Antigene suchen, in unserem Fall T-Zell-Reaktionen gegen Inselzellen Antigene sind häufig schwach 1. Bisher haben wir festgestellt, dass menschliche CD4 + T-Zell-Reaktionen zu rekombinantem Proinsulin und Glutaminsäure-Decarboxylase (GAD), Autoantigene bei Typ-1-Diabetes, nachgewiesen werden konnten mit unseren CFSE-based werdenProliferationsassay 7,8. Wir haben uns entschieden, Gewebeextrakten verwenden, um Probleme mit der Verwendung von synthetischen Peptiden 9 und 10 zugeordnet rekombinante Proteine ​​zu vermeiden. Wir wissen nicht routinemäßig hinzuzufügen Protease-Inhibitoren zu unserem Extraktionen. Die Gegenwart von Protease-Inhibitoren hemmen Antigenprozessierung und Präsentation 11 und folglich inhibieren T-Zell-Reaktionen. Stattdessen führen wir die Extraktion auf Eis in einem Versuch, Protease-vermittelten Abbau zu verhindern. Für andere Anwendungen der Einschluss von Proteaseinhibitoren kann vorteilhaft sein, wenn Proteinabbau ein Problem ist.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> 5 ml 12 x 75 mm sterile Polystyrolröhrchen </td><td> BD Falcon </td><td> 352054 </td><td></td></tr><tr><td> Caps für Rohre Polystyrolröhrchen (oben) </td><td> BD Falcon </td><td> 352032 </td><td></td></tr><tr><td> 50ml sterilen Röhrchen </td><td> Becton Dickinson </td><td> 352070 </td><td></td></tr><tr><td> Acetonitril </td><td> Mallinckradt Chemicals </td><td> 2856-10 </td><td></td></tr><tr><td> Butan-1-ol </td><td> Sigma Aldrich </td><td> 537.993-IL </td><td></td></tr><tr><td> Homogenisator: PRO200 </td><td> Bio-Strategie </td><td> 01-01200 </td><td> 10 x 115 mm Sägezahngenerator </td></tr><tr><td> Lyophilizer </td><td></td><td></td><td> Virtis, Benchtop 4K </td></tr><tr><td> Sterile Nadel 18-20 Gauge </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> Tabelle 1. Spezifisch Reagenzien und Ausrüstung.

extraction of tissue antigens for functional assays

Viele der Antigen-Targets der adaptiven Immunantwort, durch B-und T-Zellen erkannt wird, wurden nicht 1 definiert. Dies gilt insbesondere bei Autoimmunerkrankungen und Krebs 2. Unser Ziel ist es, die Antigene von menschlichen T-Zellen in der Autoimmunerkrankung Typ-1-Diabetes 1,3,4,5 erkannt zu untersuchen. Die menschliche T-Zell-Antworten gegen Gewebe, wo die Antigene durch T-Zellen erkannt werden nicht identifiziert analysieren wir ein Verfahren entwickelt, um Protein-Antigenen aus humanem Gewebe in einem Format, das kompatibel mit funktionellen Assays 6 ist extrahieren. Zuvor T-Zell-Antworten auf ungereinigten Gewebeextrakten konnte nicht gemessen werden, da die Extraktions-Verfahren liefern ein Lysat, daß Waschmittel, die toxisch für humane periphere mononukleare Zellen enthalten sein. Hier beschreiben wir ein Protokoll zur Extraktion von Proteinen aus humanen Geweben in einem Format, das nicht toxisch für humane T-Zellen. Das Gewebe wird in einem Gemisch aus Butan-1-ol, Acetonitril und homogenisiert water (BAW). Die Proteinkonzentration in der Gewebeextrakt gemessen wird und ein bekannter Masse von Protein wird in Röhrchen aliquotiert. Nach der Extraktion werden die organischen Lösungsmittel durch Lyophilisierung entfernt. Lyophilisierte Gewebeextrakten gespeichert, bis sie benötigt werden. Zur Verwendung in Tests der Immunfunktion eine Suspension von Immunzellen in geeigneten Kulturmedien, kann direkt an die lyophilisierte Extrakt zugesetzt werden. Proliferation und Zytokin-Produktion durch PBMC in Reaktion auf Extrakte hergestellt unter Verwendung dieses Verfahrens wurden leicht gemessen. Daher ermöglicht unser Verfahren die rasche Herstellung von menschlichem Gewebe-Lysaten, die als Quelle von Antigenen bei der Analyse der T-Zell-Reaktionen verwendet werden kann. Wir schlagen vor, dass diese Methode die Analyse der adaptiven Immunantwort auf Gewebe in der Transplantationsmedizin, Krebs und Autoimmunität erleichtern.

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

Extraction of Tissue Antigens for Functional Assays

Ein einfaches Protokoll zur Herstellung von menschlichem Gewebe-Extrakten, die als Quelle von Antigenen in funktioneller T-Zell-Assays verwendet werden beschrieben. Diese Methode ermöglicht es T-Zellen-Reaktionen auf Gewebe stammenden Antigenen zu messenden<em&gt; In vitro</em&gt;.

Extraktion von Tissue Antigens für funktionelle Assays

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

Nanyang Technological University

Research

JoVE Journal

Biology

10.8K Aufrufe.  St. Vincent's Institute of Medical Research. Ein einfaches Protokoll zur Herstellung von menschlichem Gewebe-Extrakten, die als Quelle von Antigenen in funktioneller T-Zell-Assays verwendet werden beschrieben. Diese Methode ermöglicht es T-Zellen-Reaktionen auf Gewebe stammenden Antigenen zu messenden In vitro.

Serie: 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.

Hell / Dunkel Transition Test für Mäuse

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, ...

Forschung

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.

Aktin Co-Sedimentation Assay; für die Analyse von Protein-Bindung an F-Aktin

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

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit Assays für Anfällen bei Epilepsie Mutanten Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

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.

Crystallizing Membranproteinen zur Strukturaufklärung mittels Lipidische Mesophasen

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

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Analyse von Schwann-Astrozyten Wechselwirkungen mit In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

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.

Pharmakologische und Functional genetischen Tests zu manipulieren Regeneration der Planarien Dugesia japonica</em

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

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Quantitativen Vergleich der Cis-Regulatory Element (CRE) Aktivitäten in transgenen Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

Zwei Arten von Assays zum Nachweis von Frog Sperm Chemotaxis

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

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.

Einsatz eines Roboters für High-Throughput-Kristallisation von Membranproteinen in Lipidische Mesophasen

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.

Small-scale Kernextrakten für funktionelle Assays von Gen-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 ...