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

Denne undersøgelse blev støttet af NIH tilskud R01HL091020, R01HL093723, R01AI077343, og R01GM096187.

1. RBC isoleret fra fuldblod <ol><li> Forbered EAS-45 løsninger. Afvejning af alle ingredienser fra tabel I og opløses i 100-200ml af DI vand. Tilsæt vand for at lave 1000ml løsning og justere pH til 8,0. Filter og alikvot af 50ml. Fryses ved -20 ° C til opbevaring. </li></ol> Bemærk: Trin 1,2 skal udføres af en uddannet læge, såsom en sygeplejerske, en institutionel Review Board godkendte protokol. <ol start="2"><li> Tegn 3-5 ml blod fra median kubiske vene i et 1...

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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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For at kunne bruge mikropipette vedhæftning frekvens-analysen bør man overveje en række vigtige skridt. Først skal du sørge for at registrere den specifikke interaktion for den receptor-ligand system af interesse. Uspecifik kontrolmålinger (jf. fig. 3, 4) sikre, at specificitet. Ideelt set bør uspecifik vedhæftning sandsynligheder være under 0,05 for al kontakt tid, varighed og til at have en betydelig forskel mellem det specifikke og uspecifikke vedhæftning sandsynligheder for hvert tidspunkt. Forskellige met...

<table border="1"><tbody><tr><th> Navn på reagenset </th><th> Firma </th><th> Katalog # </th><th> Kommentarer </th></tr><tr><td> 10x PBS </td><td> BioWhittaker </td><td> 17-517Q </td><td> Fortyndes til 1x med demineraliseret vand før brug </td></tr><tr><td> Vacutainer EDTA </td><td> BD </td><td> 366643 </td><td> RBC isolation </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> RBC isolation </td></tr><tr><td> Adenin </td><td> Sigma-Aldrich </td><td> A2786 </td><td> EAS-45 præparat </td></tr><tr><td> D-glucose (dextrose) </td><td> Sigma-Aldrich </td><td> G7528 </td><td> EAS-45 præparat </td></tr><tr><td> D-mannitol </td><td> Sigma-Aldrich </td><td> 6360 </td><td> EAS-45 præparat </td></tr><tr><td> Natriumklorid (NaCl) </td><td> Sigma-Aldrich </td><td> S7653 </td><td> EAS-45 præparat </td></tr><tr><td> Natrium, dibasisk (Na &amp; # x2082; HPO ₄) </td><td> Fisher Scientific </td><td> S374 </td><td> EAS-45 præparat </td></tr><tr><td> L-glutamin </td><td> Sigma-Aldrich </td><td> G5763 </td><td> EAS-45 præparat </td></tr><tr><td> Biotin-X-NHS </td><td> Calbiochem </td><td> 203188 </td><td> RBC biotinylation </td></tr><tr><td> Dimethylformamid (DMF) </td><td> Thermo Scientific </td><td> 20673 </td><td> RBC biotinylation </td></tr><tr><td> Borat Buffer (0.1M) </td><td> Electron Microscopy Fakultet </td><td> 11455-90 </td><td> RBC biotinylation </td></tr><tr><td> Streptavidin </td><td> Thermo Scientific </td><td> 21125 </td><td> Liganden functionalizing </td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> A0336 </td><td> Liganden functionalizing </td></tr><tr><td> Quantibrite PE perler </td><td> BD Biosciences </td><td> 340495 </td><td> Densitet kvantificering </td></tr><tr><td> Flowcytometer </td><td> BD Immunocytometry Systems </td><td> BD LSR II </td><td> Densitet mængdertificering </td></tr><tr><td> Kapillarrør 0,7 1,0 mm x 30 &quot; </td><td> Kimble Glas Inc. </td><td> 46485-1 </td><td> Mikropipette trække </td></tr><tr><td> Mineral Oil </td><td> Fisher Scientific </td><td> BP2629-1 </td><td> Afdeling samling </td></tr><tr><td> Microscope Cover Glass </td><td> Fisher Scientific </td><td> 12 til 544-G </td><td> Afdeling samling </td></tr><tr><td> PE α-menneskelige CD11a Klon HI 111 </td><td> eBioscience </td><td> 12-0119-71 </td><td> Reagens til Fig.1 </td></tr><tr><td> PE anti-humant CD54 </td><td> eBioscience </td><td> 12-0549 </td><td> Reagens til Fig.1 </td></tr><tr><td> Mouse IgG1 isotype Kontrol PE </td><td> eBioscience </td><td> 12-4714 </td><td> Reagens til Fig.1 </td></tr><tr><td> hydrauliske mikromanipulator </td><td> Narishige </td><td> MO-303 </td><td> Mikropipette systemet </td></tr><tr><td> Mekanisk manipulator </td><td> Newport </td><td> 461-xyz-m, SM-13, DM-13 </td><td> Mikropipette systemet </td></tr><tr><td> piezoelektriske oversætter </td><td> Physik Instrumente </td><td> P-840 </td><td> Mikropipette systemet </td></tr><tr><td> LabVIEW </td><td> National Instruments </td><td> Version 8.6 </td><td> Mikropipette systemet </td></tr><tr><td> DAQ bestyrelsen </td><td> National Instruments </td><td> USB-6008 </td><td> Mikropipette systemet </td></tr><tr><td> Optisk bord </td><td> Kinetics Systems </td><td> 5200-serien </td><td> Mikropipette systemet </td></tr></tbody></table>

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

Den mikropipette vedhæftning assay blev udviklet i 1998 til at måle todimensionale (2D) receptor-ligand binding kinetik 1. Analysen anvender en menneskelig røde blodlegemer (RBC) som vedhæftning sensor og præsentere celle til en af ​​de interagerende molekyler. Virksomheden beskæftiger mikromanipulering at bringe RBC i kontakt med en anden celle, der udtrykker den anden interagere molekylet med præcist kontrollerede område og tid til at gøre det muligt for obligationen dannelse. Vedhæftningen Arrangementet er detekteret som RBC forlængelse på at trække de to celler fra hinanden. Ved at styre tætheden af ​​ligander immobiliseret på RBC overfladen, er sandsynligheden for vedhæftning holdes i mid-range mellem 0 og 1. Vedhæftningen Sandsynligheden er estimeret ud fra hyppigheden af ​​friktion begivenheder i en sekvens af gentagen kontakt cyklusser mellem de to celler for en given kontakttid. Varierende kontakten tid genererer en bindende kurve. Montering af en probabilistisk model for receptor-ligand reaktionskinetik 1 til bindendekurve returnerer 2D affinitet og off-sats. Analysen er valideret ved hjælp af interaktioner mellem Fcγ receptorer med IgG Fc 1-6, selectins med glycoconjugate ligander 6-9, integriner med ligander 10-13, homotypical cadherin bindende 14, T-celle receptoren og coreceptor med peptid-dur histocompatibility komplekser 15 - 19. Metoden har været anvendt til at kvantificere reglerne i 2D kinetik af biofysiske faktorer, såsom den membran microtopology 5, membran anker 2, molekylære orientering og længde 6, transportør stivhed 9, krumning 20, og impingement kraft 20, samt biokemiske faktorer, såsom modulatorer af cytoskeleton og membran mikromiljø hvor vekselvirkende molekyler bor og overfladen organiseringen af disse molekyler 15,17,19. Metoden er også blevet brugt to undersøge den samtidige bindingen af dual-receptor-ligand arter 3,4, og trimolecular interaktioner 19 ved hjælp af en modificeret model 21. Den største fordel ved metoden er, at det giver mulighed for undersøgelse af receptorer i deres oprindelige membran miljø. Resultaterne kan være meget forskellige fra indhentet dem, der bruger renset receptorer 17. Det giver også mulighed undersøgelse af receptor-ligand interaktioner i en sub-sekunders tid med tidsmæssige opløsning langt ud over den typiske biokemiske metoder. For at illustrere mikropipette vedhæftning hyppigheden metode, vi viser kinetik måling af intercellulære adhæsionsmolekyle 1 (ICAM-1) funktionaliserede om RBC binding til integrin α L β 2 på neutrofile med dimerisk E-selektin i løsningen for at aktivere α L β 2.

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

En vedhæftning frekvens-analysen til måling af receptor-ligand interaktion kinetik, når begge molekyler er forankret på overfladen af ​​de vekselvirkende celler er beskrevet. Dette mekanisk-baserede assay er eksemplificeret ved hjælp af en mikropipette-tryk humane røde blodlegemer som vedhæftning sensor og integrin αLβ2 og intercellulære adhæsionsmolekyle-1 som vekselvirkende receptorer og ligander.

Vedhæftning Frekvens Assay for In Situ Kinetics Analyse af Cross-Junctional molekylære interaktioner i celle-celle-grænseflade

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

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

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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

Udvikling af Amelogenin-chitosan Hydrogel for<em&gt; In Vitro</em&gt; Emalje genvækst med en tæt grænseflade

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

Måling TCR-pMHC Binding<em&gt; In Situ</em&gt; Ved hjælp af et FRET-baseret assay Microscopy

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

Et tripeptid-stabiliseret Nanoemulsion af oliesyre

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

Miljømæssige dynamisk mekanisk analyse til at forudsige den blødgørende opførsel af neurale implantater

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 til kemotaktisk og molekylær analyse af human neutrofil sværmer 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-injektionsprotokol til seriel krystallografimålinger ved den australske synkrotron

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

Molekylær fjeder konstant analyse af Biomembrane Force Probe spektroskopi

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