Vedhæftning Frekvens Assay for In Situ Kinetics Analyse af Cross-Junctional molekylære interaktioner i celle-celle-grænseflade
Summary
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.
Abstract
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.
Protocol
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Denne undersøgelse blev støttet af NIH tilskud R01HL091020, R01HL093723, R01AI077343, og R01GM096187.
Materials
| Name of the reagent | Company | Catalogue # | Comments |
|---|---|---|---|
| 10x PBS | BioWhittaker | 17-517Q |
Dilute to 1x with deionized water prior to use |
| Vacutainer EDTA | BD | 366643 | RBCs isolation |
| 10ML PK100 | |||
| Histopaque 1077 | Sigma-Aldrich | 10771 | RBCs isolation |
| Adenine | Sigma-Aldrich | A2786 | EAS-45 preparation |
| D-glucose (dextrose) | Sigma-Aldrich | G7528 | EAS-45 preparation |
| D-Mannitol | Sigma-Aldrich | 6360 | EAS-45 preparation |
| Sodium Chloride (NaCl) | Sigma-Aldrich | S7653 | EAS-45 preparation |
| Sodium Phosphate, Dibasic (Na₂HPO₄) | Fisher Scientific | S374 | EAS-45 preparation |
| L-glutamine | Sigma-Aldrich | G5763 | EAS-45 preparation |
| Biotin-X-NHS | Calbiochem | 203188 | RBCs biotinylation |
| Dimethylformamide (DMF) | Thermo Scientific | 20673 | RBCs biotinylation |
| Borate Buffer (0.1M) | Electron Microscopy Sciences | 11455-90 | RBCs biotinylation |
| Streptavidin | Thermo Scientific | 21125 | Ligand functionalizing |
| BSA | Sigma-Aldrich | A0336 | Ligand functionalizing |
| Quantibrite PE Beads | BD Biosciences | 340495 | Density quantification |
| Flow cytometer | BD Immunocytometry Systems | BD LSR II |
Density quantification |
Capillary Tube 0.7-1.0mm x 30" |
Kimble Glass Inc. | 46485-1 | Micropipette pulling |
| Mineral Oil | Fisher Scientific | BP2629-1 | Chamber assembly |
| Microscope Cover Glass | Fisher Scientific | 12-544-G | Chamber assembly |
PE α-human CD11a Clone HI 111 |
eBioscience | 12-0119-71 | Reagent for Fig.1 |
| PE anti-human CD54 | eBioscience | 12-0549 | Reagent for Fig.1 |
| Mouse IgG1 Isotype Control PE | eBioscience | 12-4714 | Reagent for Fig.1 |
| hydraulic micromanipulator | Narishige | MO-303 | Micropipette system |
| Mechanical manipulator | Newport | 461-xyz-m, SM-13, DM-13 | Micropipette system |
| piezoelectric translator | Physik Instrumente | P-840 | Micropipette system |
| LabVIEW | National Instruments | Version 8.6 | Micropipette system |
| DAQ board | National Instruments | USB-6008 | Micropipette system |
| Optical table | Kinetics Systems | 5200 Series | Micropipette system |
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