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

Questo studio è stato supportato da sovvenzioni NIH R01HL091020, R01HL093723, R01AI077343 e R01GM096187.

1. Isolamento globuli rossi del sangue intero <ol><li> Preparare EAS-45 soluzioni. Pesare tutti gli ingredienti dalla tabella I e sciogliere in 100-200ml di acqua deionizzata. Aggiungere acqua per rendere la soluzione 1000ml e aggiustare il pH a 8,0. Filtro e aliquota da 50ml. Congelare a -20 ° C per la conservazione. </li></ol> Nota: Passo 1,2 dovrebbe essere eseguita da un medico addestrato ad esempio come infermiera, con un Institutional Review Board protocollo approvato. <...

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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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Per utilizzare con successo l&#39;adesione saggio micropipetta frequenza si dovrebbe considerare diversi passaggi critici. Per prima cosa, assicurarsi di registrare l&#39;interazione specifica per il recettore-ligando sistema di interesse. Misure di controllo non specifici (cfr. fig. 3, 4) garantire la specificità. Idealmente, le probabilità di adesione non specifica deve essere inferiore a 0,05 per durate di tempo tutti i contatti e di avere una significativa differenza tra le probabilità di adesione specifici e non...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Catalogo # </th><th> Commenti </th></tr><tr><td> 10x PBS </td><td> BioWhittaker </td><td> 17-517Q </td><td> Diluire a 1x con acqua deionizzata prima dell&#39;uso </td></tr><tr><td> Vacutainer EDTA </td><td> BD </td><td> 366643 </td><td> RBC isolamento </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 isolamento </td></tr><tr><td> Adenina </td><td> Sigma-Aldrich </td><td> A2786 </td><td> EAS-45 preparazione </td></tr><tr><td> D-glucosio (destrosio) </td><td> Sigma-Aldrich </td><td> G7528 </td><td> EAS-45 preparazione </td></tr><tr><td> D-mannitolo </td><td> Sigma-Aldrich </td><td> 6360 </td><td> EAS-45 preparazione </td></tr><tr><td> Cloruro di sodio (NaCl) </td><td> Sigma-Aldrich </td><td> S7653 </td><td> EAS-45 preparazione </td></tr><tr><td> Fosfato di sodio, bibasico (Na &amp; # x2082; HPO ₄) </td><td> Fisher Scientific </td><td> S374 </td><td> EAS-45 preparazione </td></tr><tr><td> L-glutammina </td><td> Sigma-Aldrich </td><td> G5763 </td><td> EAS-45 preparazione </td></tr><tr><td> Biotina-X-NHS </td><td> Calbiochem </td><td> 203188 </td><td> RBC biotinilazione </td></tr><tr><td> Dimetilformammide (DMF) </td><td> Thermo Scientific </td><td> 20673 </td><td> RBC biotinilazione </td></tr><tr><td> Borato Buffer (0,1 M) </td><td> Electron Microscopy Sciences </td><td> 11455-90 </td><td> RBC biotinilazione </td></tr><tr><td> Streptavidina </td><td> Thermo Scientific </td><td> 21125 </td><td> Ligand funzionalizzazione </td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> A0336 </td><td> Ligand funzionalizzazione </td></tr><tr><td> Quantibrite PE Perline </td><td> BD Biosciences </td><td> 340495 </td><td> Densità di quantificazione </td></tr><tr><td> Citometro a flusso </td><td> BD Immunocytometry Sistemi </td><td> BD LSR II </td><td> Densità di quanidentificazione </td></tr><tr><td> Tubo capillare 0,7-1.0mm x 30 &quot; </td><td> Kimble Glass Inc. </td><td> 46485-1 </td><td> Micropipetta tirando </td></tr><tr><td> Olio minerale </td><td> Fisher Scientific </td><td> BP2629-1 </td><td> Camera di montaggio </td></tr><tr><td> Microscopio copertura in vetro </td><td> Fisher Scientific </td><td> 12-544-G </td><td> Camera di montaggio </td></tr><tr><td> PE-α umano CD11a Clone HI 111 </td><td> eBioscience </td><td> 12-0119-71 </td><td> Reagente per Fig.1 </td></tr><tr><td> PE anti-CD54 umano </td><td> eBioscience </td><td> 12-0549 </td><td> Reagente per Fig.1 </td></tr><tr><td> IgG1 Isotipo controllo PE </td><td> eBioscience </td><td> 12-4714 </td><td> Reagente per Fig.1 </td></tr><tr><td> micromanipolatore idraulico </td><td> Narishige </td><td> MO-303 </td><td> Micropipetta sistema </td></tr><tr><td> Manipolatore meccanico </td><td> Newport </td><td> 461-xyz-m, SM-13, DM-13 </td><td> Micropipetta sistema </td></tr><tr><td> traduttore piezoelettrico </td><td> Physik Instrumente </td><td> P-840 </td><td> Micropipetta sistema </td></tr><tr><td> LabVIEW </td><td> National Instruments </td><td> Versione 8.6 </td><td> Micropipetta sistema </td></tr><tr><td> Scheda DAQ </td><td> National Instruments </td><td> USB-6008 </td><td> Micropipetta sistema </td></tr><tr><td> Tavolo ottico </td><td> Cinetica Sistemi </td><td> Serie 5200 </td><td> Micropipetta sistema </td></tr></tbody></table>

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

Il test di adesione micropipetta è stato sviluppato nel 1998 per misurare bidimensionali (2D), recettore-ligando cinetica di legame 1. Il test utilizza un globulo rosso umano (RBC) come sensore di adesione e delle cellule che si presentano per una delle molecole interagenti. Impiega micromanipolazione per portare il RBC in contatto con un&#39;altra cellula che esprime l&#39;altra molecola che interagisce con zona controllata con precisione e tempo per consentire la formazione di legame. L&#39;evento viene rilevato come l&#39;adesione allungamento RBC su tirando le due celle di distanza. Controllando la densità dei ligandi immobilizzato sulla superficie dei globuli rossi, la probabilità di adesione è tenuto in mid-range compreso tra 0 e 1. La probabilità di adesione è stimata dalla frequenza di eventi adesione in una sequenza di cicli ripetuti contatti tra le due cellule per un dato tempo di contatto. Variando il tempo di contatto genera una curva vincolante. Montaggio di un modello probabilistico per recettore-ligando cinetica di reazione 1 al legamecurva restituisce l&#39;affinità 2D e off-rate. Il test è stato validato mediante interazioni dei recettori Fcy con IgG Fc 1-6, selectine con leganti glicoconiugato 6-9, integrine con leganti 10-13, homotypical vincolante caderina 14, recettore delle cellule T e corecettore con peptide-grandi complessi di istocompatibilità 15 - 19. Il metodo è stato utilizzato per quantificare regolamenti della cinetica 2D da fattori biofisici, come ad esempio la membrana microtopology 5, della membrana di ancoraggio 2, orientamento molecolare e lunghezza 6, rigidità vettore 9, curvatura 20, e la forza impingement 20, così come i fattori biochimici, come modulatori del microambiente citoscheletro e membrana in cui le molecole interagiscono risiedono e l&#39;organizzazione della superficie di queste molecole 15,17,19. Il metodo è stato utilizzato anche to studiare il legame simultaneo di doppio recettore-ligando specie 3,4, e le interazioni trimolecular 19 con un modello modificato 21. Il principale vantaggio del metodo è che permette lo studio dei recettori di membrana nel loro ambiente nativo. I risultati potrebbero essere molto diversi da quelli ottenuti con recettori purificati 17. Consente inoltre lo studio del recettore-ligando interazioni in un lasso di tempo inferiori al secondo con risoluzione temporale ben oltre i tipici metodi biochimici. Per illustrare l&#39;adesione metodo micropipetta frequenza, ci mostrano la misurazione cinetica della molecola di adesione intercellulare 1 (ICAM-1) funzionalizzati su globuli rossi legame β integrina α L 2 sui neutrofili con dimerica E-selectina nella soluzione per attivare α β L 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

Un test per la misurazione della frequenza adesione recettore-ligando cinetiche di interazione quando entrambe le molecole sono ancorate sulla superficie delle cellule interagiscono è descritto. Questo test meccanico-based è esemplificato con una micropipetta pressurizzato globulo rosso umano come sensore di adesione e integrina αLβ2 e molecola di adesione intercellulare-1 come interagire recettori e ligandi.

Adesione saggio di frequenza per In Situ Analisi cinetica delle interazioni molecolari incrociati giunzionale alla cellula-cellula di interfaccia

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

Sviluppo di amelogenina-chitosano Hydrogel per<em&gt; In Vitro</em&gt; Smalto ricrescita con una interfaccia Dense

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

Misurazione TCR-pMHC Binding<em&gt; In Situ</em&gt; Utilizzando un microscopio saggio basato FRET-

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

Nanoemulsione Tripeptide-stabilizzato di acido oleico

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

Analisi meccanica dinamica ambientale per predire il comportamento di rammollimento di impianti neurali

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

Microarray di bioparticelle per l'analisi chemiotattica e molecolare dello sciame neutrofilo umano 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

Protocollo di iniezione lipidico per le misurazioni della cristallografia seriale presso il sincrotrone australiano

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

Analisi della costante molecolare della molla mediante spettroscopia a sonda a forza biomembrana

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