Name: adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface
Uploaded: 2011-11-02T13:00:00
Description: Watch this Scientific Journal Video about Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface at JoVE.com

Diese Studie wurde vom NIH gewährt R01HL091020, R01HL093723, R01AI077343 und R01GM096187 unterstützt.

1. RBCs Isolierung aus Vollblut <ol><li> Bereiten Sie EAS-45-Lösungen. Wägen Sie alle Zutaten aus Tabelle I und lösen sich in 100-200ml VE-Wasser. Wasser hinzu, bis 1000ml Lösung zu machen und pH-Wert auf 8,0. Filter und Aliquot von 50ml. Einfrieren bei -20 ° C für die Lagerung. </li></ol> Hinweis: Schritt 1.2 sollte durch einen geschulten Arzt, wie eine Krankenschwester durchgeführt werden, mit einem Institutional Review Board genehmigten Protokoll. <ol start="2"><l...

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	<li>Chesla, S. E., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+two-dimensional+receptor-ligand+binding+kinetics+by+micropipette.">Measuring two-dimensional receptor-ligand binding kinetics by micropipette.</a> Biophys. J. 75, 1553-1572 (1998).</li><li>Chesla, S. E., Li, P., Nagarajan, S., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane+anchor+influences+ligand+binding+two-dimensional+kinetic+rates+and+three-dimensional+affinity+of+FcgammaRIII+(CD16).">The membrane anchor influences ligand binding two-dimensional kinetic rates and three-dimensional affinity of FcgammaRIII (CD16).</a> J. Biol. Chem. 275, 10235-10246 (2000).</li><li>Williams, T. 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Um erfolgreich mit der Mikropipette Haftung Frequenz Assay man mehrere kritische Schritte in Betracht ziehen sollten. Stellen Sie zunächst sicher, um die spezifische Wechselwirkung für die Rezeptor-Ligand-System von Interesse aufnehmen. Unspezifische Kontrollmessungen (vgl. Abb.. 3, 4) gewährleisten die Spezifität. Idealerweise sollten unspezifische Adhäsion Wahrscheinlichkeiten unter 0,05 für jeden Kontakt Zeitdauern zu sein und einen signifikanten Unterschied zwischen der spezifischen und unspezifischen Haftung ...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog # </th><th> Kommentare </th></tr><tr><td> 10x PBS </td><td> BioWhittaker </td><td> 17-517Q </td><td> Verdünnen auf 1x mit VE-Wasser vor dem Gebrauch </td></tr><tr><td> Vacutainer EDTA </td><td> BD </td><td> 366643 </td><td> Erythrozyten isoliert </td></tr><tr><td> 10ML PK100 </td><td></td><td></td><td></td></tr><tr><td> Histopaque 1077 </td><td> Sigma-Aldrich </td><td> 10771 </td><td> Erythrozyten isoliert </td></tr><tr><td> Adenin </td><td> Sigma-Aldrich </td><td> A2786 </td><td> EAS-45 Vorbereitung </td></tr><tr><td> D-Glucose (Dextrose) </td><td> Sigma-Aldrich </td><td> G7528 </td><td> EAS-45 Vorbereitung </td></tr><tr><td> D-Mannitol </td><td> Sigma-Aldrich </td><td> 6360 </td><td> EAS-45 Vorbereitung </td></tr><tr><td> Natriumchlorid (NaCl) </td><td> Sigma-Aldrich </td><td> S7653 </td><td> EAS-45 Vorbereitung </td></tr><tr><td> Sodium Phosphate, Dibasic (Na &amp; # x2082; HPO ₄) </td><td> Fisher Scientific </td><td> S374 </td><td> EAS-45 Vorbereitung </td></tr><tr><td> L-Glutamin </td><td> Sigma-Aldrich </td><td> G5763 </td><td> EAS-45 Vorbereitung </td></tr><tr><td> Biotin-X-NHS </td><td> Calbiochem </td><td> 203188 </td><td> RBCs Biotinylierung </td></tr><tr><td> Dimethylformamid (DMF) </td><td> Thermo Scientific </td><td> 20673 </td><td> RBCs Biotinylierung </td></tr><tr><td> Boratpuffer (0,1 M) </td><td> Electron Microscopy Sciences </td><td> 11455-90 </td><td> RBCs Biotinylierung </td></tr><tr><td> Streptavidin </td><td> Thermo Scientific </td><td> 21125 </td><td> Ligand Funktionalisierung </td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> A0336 </td><td> Ligand Funktionalisierung </td></tr><tr><td> Quantibrite PE Beads </td><td> BD Biosciences </td><td> 340495 </td><td> Dichte Quantifizierung </td></tr><tr><td> Durchflusszytometer </td><td> BD Immunocytometry Systems </td><td> BD LSR II </td><td> Dichte quantification </td></tr><tr><td> Kapillarrohr 0,7-1.0mm x 30 &quot; </td><td> Kimble Glass Inc. </td><td> 46485-1 </td><td> Micropipette ziehen </td></tr><tr><td> Mineralöl </td><td> Fisher Scientific </td><td> BP2629-1 </td><td> Kammeranordnung </td></tr><tr><td> Mikroskop Deckglas </td><td> Fisher Scientific </td><td> 12 bis 544-G </td><td> Kammeranordnung </td></tr><tr><td> PE α-human CD11a Clone HALLO 111 </td><td> eBioscience </td><td> 12-0119-71 </td><td> Reagenz für Abb.1 </td></tr><tr><td> PE anti-human CD54 </td><td> eBioscience </td><td> 12-0549 </td><td> Reagenz für Abb.1 </td></tr><tr><td> Maus IgG1 Isotyp-Kontroll-PE </td><td> eBioscience </td><td> 12-4714 </td><td> Reagenz für Abb.1 </td></tr><tr><td> hydraulischen Mikromanipulator </td><td> Narishige </td><td> MO-303 </td><td> Micropipette System </td></tr><tr><td> Mechanische Manipulator </td><td> Newport </td><td> 461-xyz-m, SM-13, DM-13 </td><td> Micropipette System </td></tr><tr><td> piezoelektrische Übersetzer </td><td> Physik Instrumente </td><td> P-840 </td><td> Micropipette System </td></tr><tr><td> LabVIEW </td><td> National Instruments </td><td> Version 8.6 </td><td> Micropipette System </td></tr><tr><td> DAQ-Karte </td><td> National Instruments </td><td> USB-6008 </td><td> Micropipette System </td></tr><tr><td> Optische Tisch </td><td> Kinetics Systeme </td><td> 5200 Series </td><td> Micropipette System </td></tr></tbody></table>

adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface

Die Mikropipette Adhäsionsassay wurde 1998 entwickelt, um zweidimensionale (2D)-Rezeptor-Ligand-Bindung Kinetik 1 zu messen. Der Test verwendet eine menschliche rote Blutzellen (RBC) als Haftvermittler Sensor und präsentierenden Zelle für eine der wechselwirkenden Moleküle. Das Unternehmen beschäftigt Mikromanipulation der RBC in Kontakt mit einer anderen Zelle, dass die anderen interagieren Molekül drückt mit präzise kontrollierten Raum und Zeit, um Bindung zu ermöglichen bringen. Die Haftung Veranstaltung ist als RBC Dehnung beim Ziehen der beiden Zellen voneinander entdeckt. Durch die Steuerung der Dichte des Liganden auf der RBC Oberfläche immobilisiert, ist die Wahrscheinlichkeit der Adhäsion in Mid-Range zwischen 0 und 1 gehalten. Die Haftung Wahrscheinlichkeit ist von der Frequenz der Haftung Ereignisse in eine Folge von wiederholtem Kontakt Zyklen zwischen den beiden Zellen für eine bestimmte Einwirkzeit geschätzt. Durch Variation der Einwirkzeit erzeugt eine verbindliche Kurve. Der Einbau einer probabilistischen Modell für die Rezeptor-Ligand-Reaktionskinetik 1, um die BindungKurve gibt die 2D-Affinität und off-Rate. Der Test wurde validiert mit Wechselwirkungen von Fcy Rezeptoren mit IgG Fc 1-6, Selektine mit Glykokonjugat Liganden 6-9, Integrine mit Liganden 10-13, homotypical Cadherin binden 14, T-Zell-Rezeptor und Korezeptor mit Peptid-MHC-Komplexe 15 - 19. Die Methode wurde verwendet, um Vorschriften von 2D-Kinetik von biophysikalischen Faktoren, wie die Membran microtopology 5, Membrananker 2, molekulare Orientierung und Länge 6, Träger Steifigkeit 9, Krümmung 20 und Impingement Kraft 20, sowie biochemische Faktoren zu quantifizieren, wie Modulatoren des Zytoskeletts und Membranmikroumgebung wo die interagierende Moleküle befinden und die Oberfläche Organisation dieser Moleküle 15,17,19. Die Methode wurde auch t verwendeto Studie der gleichzeitigen Bindung von zwei Rezeptor-Ligand-Arten 3,4 und trimolekulare Interaktionen 19 mit einem modifizierten Modell 21. Der große Vorteil der Methode ist, dass es zu studieren von Rezeptoren in ihrer Muttersprache Membran-Umgebung ermöglicht. Die Ergebnisse könnten erheblich von denen unter Verwendung gereinigter Rezeptoren 17. Es ermöglicht auch Erforschung der Rezeptor-Ligand-Wechselwirkungen in einem Sub-Sekunden-Zeitskala mit zeitlicher Auflösung die weit über die typischen biochemischen Methoden. Zur Veranschaulichung der Mikropipette Haftung Frequenz-Methode zeigen wir, Kinetik Messung von ICAM 1 (ICAM-1) auf Erythrozyten Bindung an Integrin α L β 2 auf Neutrophilen mit dimeren E-Selektin in die Lösung α L β 2 aktivieren funktionalisiert.

Watch this Scientific Journal Video about Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface at JoVE.com

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

Eine Haftung Frequenz-Assay zur Messung der Rezeptor-Ligand-Wechselwirkung Kinetik, wenn beide Moleküle auf der Oberfläche der interagierenden Zellen verankert sind beschrieben. Diese mechanisch-basierter Assay wird beispielhaft mit einer Mikropipette unter Druck menschlichen roten Blutkörperchen als Haftvermittler Sensor und Integrin αLβ2 und ICAM-1 als interagierende Rezeptoren und Liganden.

Adhesion Frequency-Assay für In Situ Kinetics Analyse der Cross-Junktionaler Molecular Interactions an der Zell-Zell-Schnittstelle

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Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Bestimmung der Transport von Xenobiotika und Nanomaterialien über die Plazenta mit dem<em&gt; Ex vivo</em&gt; Menschliche Plazentaperfusion Modell

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Entwicklung von Amelogenin-Chitosan-Hydrogel für<em&gt; In-vitro-</em&gt; Emaille Nachwachsen mit einem dichten Schnittstelle

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

Mess TCR-pMHC Binding<em&gt; In-situ-</em&gt; Mit einem FRET-Mikroskopie Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

Ein Tripeptid-stabilisierten Nanoemulsion Ölsäure

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

Ökologische dynamische mechanische Analyse um die Erweichung Verhalten der neuronale Implantate

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Biopartikel-Mikroarrays für chemotaktische und molekulare Analysen des menschlichen Neutrophilen-Schwärmens in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

Lipidico Injektionsprotokoll für serielle Kristallographiemessungen am australischen Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

Molekulare Federkonstantenanalyse mittels Biomembran-Kraftsondenspektroskopie

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...