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

Denna studie fick stöd av NIH bidrag R01HL091020, R01HL093723, R01AI077343 och R01GM096187.

1. RBC: er isolering från helblod <ol><li> Förbered EAS-45-lösningar. Väg upp alla ingredienser från tabell I och lös upp i 100-200ml av DI-vatten. Tillsätt vatten till 1000 ml lösning och justera pH till 8,0. Filter och delmängd av 50ml. Frys vid -20 ° C för förvaring. </li></ol> OBS: Steg 1,2 bör utföras av en utbildad läkare eller sjuksköterska som en sjuksköterska, med en granskningsnämnder godkänt protokoll. <ol start="2"><li> Rita 3-5 ml blod från ...

<ol>
	<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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För att framgångsrikt använda mikropipett analysen vidhäftning frekvens man bör tänka på flera viktiga steg. Börja med att kontrollera att spela in den specifika interaktionen för receptor-ligand systemet av intresse. Ospecifik kontrollmätningar (jfr figur. 3, 4) se till att specificitet. Helst bör ospecifik vidhäftning sannolikheter ligga under 0,05 för samtliga löptider kontakttid och att ha en betydande skillnad mellan det specifika och ospecifika vidhäftning sannolikheter för varje tidpunkt. Olika me...

<table border="1"><tbody><tr><th> Reagensets namn </th><th> Företag </th><th> Catalogue # </th><th> Kommentarer </th></tr><tr><td> 10x PBS </td><td> BioWhittaker </td><td> 17-517Q </td><td> Späd till 1x med avjoniserat vatten före användning </td></tr><tr><td> Vacutainer EDTA </td><td> BD </td><td> 366.643 </td><td> Röda blodkropparna isolering </td></tr><tr><td> 10ml PK100 </td><td></td><td></td><td></td></tr><tr><td> Histopaque 1077 </td><td> Sigma-Aldrich </td><td> 10.771 </td><td> Röda blodkropparna isolering </td></tr><tr><td> Adenin </td><td> Sigma-Aldrich </td><td> A2786 </td><td> EAS-45 preparatet </td></tr><tr><td> D-glukos (dextros) </td><td> Sigma-Aldrich </td><td> G7528 </td><td> EAS-45 preparatet </td></tr><tr><td> D-Mannitol </td><td> Sigma-Aldrich </td><td> 6360 </td><td> EAS-45 preparatet </td></tr><tr><td> Natriumklorid (NaCl) </td><td> Sigma-Aldrich </td><td> S7653 </td><td> EAS-45 preparatet </td></tr><tr><td> Natriumfosfat, dibasiskt (Na &amp; # x2082, HPO ₄) </td><td> Fisher Scientific </td><td> S374 </td><td> EAS-45 preparatet </td></tr><tr><td> L-glutamin </td><td> Sigma-Aldrich </td><td> G5763 </td><td> EAS-45 preparatet </td></tr><tr><td> Biotin-X-NHS </td><td> Calbiochem </td><td> 203.188 </td><td> Röda blodkropparna biotinylation </td></tr><tr><td> Dimetylformamid (DMF) </td><td> Thermo Scientific </td><td> 20.673 </td><td> Röda blodkropparna biotinylation </td></tr><tr><td> Boratbuffert (0,1 miljoner) </td><td> Elektronmikroskopi Sciences </td><td> 11.455-90 </td><td> Röda blodkropparna biotinylation </td></tr><tr><td> Streptavidin </td><td> Thermo Scientific </td><td> 21.125 </td><td> Ligand functionalizing </td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> A0336 </td><td> Ligand functionalizing </td></tr><tr><td> Quantibrite PE Pärlor </td><td> BD Biosciences </td><td> 340.495 </td><td> Densitet kvantifiering </td></tr><tr><td> Flödescytometer </td><td> BD Immunocytometry Systems </td><td> BD LSR II </td><td> Densitet Quantifiering </td></tr><tr><td> Kapillärrör 0.7-1.0mm x 30 &quot; </td><td> Kimble Glas Inc. </td><td> 46.485-1 </td><td> Mikropipett dra </td></tr><tr><td> Mineralolja </td><td> Fisher Scientific </td><td> BP2629-1 </td><td> Avdelningen montering </td></tr><tr><td> Mikroskop skyddsglas </td><td> Fisher Scientific </td><td> 12-544-G </td><td> Avdelningen montering </td></tr><tr><td> PE α-mänskliga CD11a Clone HI 111 </td><td> eBioscience </td><td> 12-0119-71 </td><td> Reagens för Fig.1 </td></tr><tr><td> PE anti-humant CD54 </td><td> eBioscience </td><td> 12-0549 </td><td> Reagens för Fig.1 </td></tr><tr><td> Mouse IgG1 isotypisk kontroll PE </td><td> eBioscience </td><td> 12-4714 </td><td> Reagens för Fig.1 </td></tr><tr><td> hydraulisk mikromanipulator </td><td> Narishige </td><td> MO-303 </td><td> Mikropipett systemet </td></tr><tr><td> Mekanisk manipulator </td><td> Newport </td><td> 461-xyz-M, SM-13, DM-13 </td><td> Mikropipett systemet </td></tr><tr><td> piezoelektriska översättare </td><td> Physik Instrumente </td><td> P-840 </td><td> Mikropipett systemet </td></tr><tr><td> LabVIEW </td><td> National Instruments </td><td> Version 8.6 </td><td> Mikropipett systemet </td></tr><tr><td> DAQ ombord </td><td> National Instruments </td><td> USB-6008 </td><td> Mikropipett systemet </td></tr><tr><td> Optiska bord </td><td> Kinetics Systems </td><td> 5200-serien </td><td> Mikropipett systemet </td></tr></tbody></table>

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

Den mikropipett vidhäftning test utvecklades 1998 för att mäta tvådimensionella (2D) receptor-ligand bindning kinetik 1. Analysen använder en mänsklig röda blodkroppar (RBC) som vidhäftning sensor och presentera cell för en av de samverkande molekyler. Den sysselsätter mikromanipulation att få RBC i kontakt med en annan cell som uttrycker det andra interagera molekyl med exakt kontrollerat område och tid att band bildas. Vidhäftningen händelsen detekteras som RBC töjning på att dra i två celler isär. Genom att kontrollera tätheten av ligander immobiliserade på RBC ytan, är sannolikheten för vidhäftning hålls i mitten av intervallet mellan 0 och 1. Vidhäftningen Sannolikheten beräknas från frekvensen vidhäftning händelser i en sekvens av upprepad kontakt cykler mellan två celler för en viss kontakttid. Varierande kontakttiden genererar ett bindande kurva. Montering av en probabilistisk modell för receptor-ligand reaktionskinetik 1 till bindandekurvan återgår 2D affinitet och off-takt. Analysen har validerats med hjälp av interaktioner mellan Fcγ receptorer med IgG Fc 1-6, selectins med glycoconjugate ligander 6-9, integriner med ligander 10-13, homotypical cadherin bindande 14, T cells receptor och coreceptor med peptid-dur histocompatibility komplex 15 - 19. Metoden har använts för att kvantifiera föreskrifter 2D kinetik av biofysiska faktorer, som membranet microtopology 5, membran ankare 2, molekylär inriktning och längd 6, bärare stelhet 9, krökning 20 och impingement kraft 20 samt biokemiska faktorer, som modulatorer av cytoskelettet och membran mikromiljö där interagerande molekyler vistas och ytan organisationen av dessa molekyler 15,17,19. Metoden har också använts to studerar samtidigt bindning av dubbla receptor-ligand arter 3,4 och trimolecular interaktioner 19 med hjälp av en modifierad modell 21. Den stora fördelen med metoden är att det tillåter studier av receptorer i sitt eget membran miljö. Resultaten kan vara mycket olika de som erhålls med renade receptorer 17. Det ger också studiet av receptor-ligand interaktioner i en sub-sekunders tid med tidsupplösning långt utanför den typiska biokemiska metoder. För att illustrera mikropipett metoden vidhäftning frekvens visar vi kinetik mätning av intercellulär adhesionsmolekyl 1 (ICAM-1) functionalized på röda blodkroppar bindning till integrin α L β 2 på neutrofiler med dimeriskt E-selektin i lösningen för att aktivera α L β 2.

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

En vidhäftning frekvens test för mätning av receptor-ligand interaktioner kinetik när båda molekyler är förankrade på ytan av de samverkande celler beskrivs. Denna mekaniskt baserad analys exemplifieras med hjälp av en mikropipett trycksatt människans röda blodkroppar som adhesion sensor och integrin αLβ2 och intercellulära adhesionsmolekyl-1 som interagerande receptorer och ligander.

Vidhäftning Frekvens analys för In Situ Kinetik Analys av Cross-Junktional Molekylär växelverkan vid cell-cell Interface

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

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

Utveckling av Amelogenin-chitosan Hydrogel för<em&gt; In Vitro</em&gt; Emalj Återväxt med en Tät gränssnitt

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

Mätt TCR-pMHC Bindning<em&gt; In Situ</em&gt; Med en FRET-baserad Mikroskopi analys

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

En tripeptid-stabiliserad Nanoemulsion oljesyra

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

Dynamiska mekaniska miljöanalys att förutsäga beteendet mjukgörande med neurala implantat

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 Mikroarrayer för chemotaktisk och molekylär analys av human neutrophil svärma 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 Injection Protocol för seriella kristallografimätningar vid den australiska synkrotronen

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

Molekylär vårkonstant analys av biomembrankraftsondspektroskopi

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