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

Deze studie werd ondersteund door de NIH subsidie ​​R01HL091020, R01HL093723, R01AI077343, en R01GM096187.

1. RBC isolatie van de volbloed <ol><li> Bereid EAS-45 oplossingen. Afweging van alle ingrediënten uit Tabel I en los op in 100-200ml van DI water. Voeg water toe om 1000ml oplossing te maken en de pH aan te passen aan 8,0. Filter en aliquot van 50ml. Invriezen bij -20 ° C voor opslag. </li></ol> Opmerking: Stap 1.2 moet worden uitgevoerd door een opgeleide medische professional, zoals een verpleegkundige, met een Institutional Review Board goedgekeurde protocol. <ol star...

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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. E., 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=Concurrent+and+independent+binding+of+Fcgamma+receptors+IIa+and+IIIb+to+surface-bound+IgG.">Concurrent and independent binding of Fcgamma receptors IIa and IIIb to surface-bound IgG.</a> Biophys. J. 79, 1867-1875 (2000).</li><li>Williams, T. 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=Concurrent+binding+to+multiple+ligands:+kinetic+rates+of+CD16b+for+membrane-bound+IgG1+and+IgG2.">Concurrent binding to multiple ligands: kinetic rates of CD16b for membrane-bound IgG1 and IgG2.</a> Biophys. J. 79, 1858-1866 (2000).</li><li>Williams, T. E., 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=Quantifying+the+impact+of+membrane+microtopology+on+effective+two-dimensional+affinity.">Quantifying the impact of membrane microtopology on effective two-dimensional affinity.</a> J. Biol. Chem. 276, 13283-13288 (2001).</li><li>Huang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+effects+of+molecular+orientation+and+length+on+two-dimensional+receptor-ligand+binding+kinetics.">Quantifying the effects of molecular orientation and length on two-dimensional receptor-ligand binding kinetics.</a> J. Biol. Chem. 279, 44915-44923 (2004).</li><li>Long, M., Zhao, H., Huang, K. S., 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=Kinetic+measurements+of+cell+surface+E-selectin/carbohydrate+ligand+interactions.">Kinetic measurements of cell surface E-selectin/carbohydrate ligand interactions.</a> Ann. Biomed. Eng. 29, 935-946 (2001).</li><li>Chen, W., Evans, E. A., McEver, R. 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=Monitoring+receptor-ligand+interactions+between+surfaces+by+thermal+fluctuations.">Monitoring receptor-ligand interactions between surfaces by thermal fluctuations.</a> Biophys. J. 94, 694-701 (2008).</li><li>Wu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+carrier+stiffness+and+microtopology+on+two-dimensional+kinetics+of+P-selectin+and+P-selectin+glycoprotein+ligand-1+(PSGL-1)+interactions.">Impact of carrier stiffness and microtopology on two-dimensional kinetics of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) interactions.</a> J. Biol. Chem. 282, 9846-9854 (2007).</li><li>Waugh, R. E., Lomakina, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+site+formation,+not+bond+kinetics,+limits+adhesion+rate+between+human+neutrophils+and+immobilized+vascular+cell+adhesion+molecule+1.">Active site formation, not bond kinetics, limits adhesion rate between human neutrophils and immobilized vascular cell adhesion molecule 1.</a> Biophys. J. 96, 268-275 (2009).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+kinetics+regulation+of+alphaLbeta2-ICAM-1+interaction+by+conformational+changes+of+the+alphaL-inserted+domain.">Two-dimensional kinetics regulation of alphaLbeta2-ICAM-1 interaction by conformational changes of the alphaL-inserted domain.</a> J. Biol. Chem. 280, 42207-42218 (2005).</li><li>Lomakina, E. B., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+between+human+neutrophils+and+immobilized+endothelial+ligand+vascular+cell+adhesion+molecule+1:+divalent+ion+effects.">Adhesion between human neutrophils and immobilized endothelial ligand vascular cell adhesion molecule 1: divalent ion effects.</a> Biophys. J. 96, 276-284 (2009).</li><li>Chen, W., Lou, J., 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=Forcing+switch+from+short-+to+intermediate-+and+long-lived+states+of+the+alphaA+domain+generates+LFA-1/ICAM-1+catch+bonds.">Forcing switch from short- to intermediate- and long-lived states of the alphaA domain generates LFA-1/ICAM-1 catch bonds.</a> J. Biol. Chem. 285, 35967-35978 (2010).</li><li>Chien, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+stage+cadherin+kinetics+require+multiple+extracellular+domains+but+not+the+cytoplasmic+region.">Two stage cadherin kinetics require multiple extracellular domains but not the cytoplasmic region.</a> J. Biol. Chem. 283, 1848-1856 (2008).</li><li>Huang, J., Edwards, L. J., Evavold, B. D., 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=Kinetics+of+MHC-CD8+interaction+at+the+T+cell+membrane.">Kinetics of MHC-CD8 interaction at the T cell membrane.</a> J. Immunol. 179, 7653-7662 (2007).</li><li>Wasserman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+variant+peptide-mediated+anergy+of+encephalitogenic+T+cells+requires+SHP-1.">MHC variant peptide-mediated anergy of encephalitogenic T cells requires SHP-1.</a> J. Immunol. 181, 6843-6849 (2008).</li><li>Huang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinetics+of+two-dimensional+TCR+and+pMHC+interactions+determine+T-cell+responsiveness.">The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness.</a> Nature. 464, 932-936 .</li><li>Sabatino, J. J., Huang, J., Zhu, C., Evavold, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+prevalence+of+low+affinity+peptide-MHC+II+tetramer-negative+effectors+during+polyclonal+CD4++T+cell+responses.">High prevalence of low affinity peptide-MHC II tetramer-negative effectors during polyclonal CD4+ T cell responses.</a> J. Exp. Med. 208, 81-90 (2011).</li><li>Jiang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-stage+cooperative+T+cell+receptor-peptide+major+histocompatibility+complex-CD8+trimolecular+interactions+amplify+antigen+discrimination.">Two-stage cooperative T cell receptor-peptide major histocompatibility complex-CD8 trimolecular interactions amplify antigen discrimination.</a> Immunity. 34, 13-23 (2011).</li><li>Spillmann, C. M., Lomakina, E., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesive+contact+dependence+on+impingement+force.">Neutrophil adhesive contact dependence on impingement force.</a> Biophys. J. 87, 4237-4245 (2004).</li><li>Zhu, C., Williams, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+concurrent+binding+of+multiple+molecular+species+in+cell+adhesion.">Modeling concurrent binding of multiple molecular species in cell adhesion.</a> Biophys. J. 79, 1850-1857 (2000).</li><li>Downey, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retention+of+leukocytes+in+capillaries:+role+of+cell+size+and+deformability.">Retention of leukocytes in capillaries: role of cell size and deformability.</a> J. Appl. Physiol. 69, 1767-1778 (1990).</li><li>Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+and+kinetic+analysis+of+Fcgamma+receptor+IIIa+(CD16a)+binding+to+IgG+ligands.">Affinity and kinetic analysis of Fcgamma receptor IIIa (CD16a) binding to IgG ligands.</a> J. Biol. Chem. 282, 6210-6221 (2007).</li><li>Chen, W., Zarnitsyna, V. I., Sarangapani, K. K., Huang, J., 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+Receptor-Ligand+Binding+Kinetics+on+Cell+Surfaces:+From+Adhesion+Frequency+to+Thermal+Fluctuation+Methods.">Measuring Receptor-Ligand Binding Kinetics on Cell Surfaces: From Adhesion Frequency to Thermal Fluctuation Methods.</a> Cell. Mol. Bioeng. 1, 276-288 (2008).</li><li>Thoumine, O., Kocian, P., Kottelat, A., Meister, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+binding+of+fibroblasts+to+fibronectin:+optical+tweezers+experiments+and+probabilistic+analysis.">Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis.</a> Eur. Biophys. J. 29, 398-408 (2000).</li><li>Ounkomol, C., Xie, H., Dayton, P. A., Heinrich, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+horizontal+force+probe+for+mechanical+tests+on+pipette-held+cells,+particles,+and+membrane+capsules.">Versatile horizontal force probe for mechanical tests on pipette-held cells, particles, and membrane capsules.</a> Biophys. J. 96, 1218-1231 (2009).</li><li>Piper, J. W., Swerlick, R. A., 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=Determining+force+dependence+of+two-dimensional+receptor-ligand+binding+affinity+by+centrifugation.">Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation.</a> Biophys. J. 74, 492-513 (1998).</li><li>Li, P., 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=Analysis+of+competition+binding+between+soluble+and+membrane-bound+ligands+for+cell+surface+receptors.">Analysis of competition binding between soluble and membrane-bound ligands for cell surface receptors.</a> Biophys. J. 77, 3394-3406 (1999).</li><li>Long, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probabilistic+modeling+of+rosette+formation.">Probabilistic modeling of rosette formation.</a> Biophys. J. 91, 352-363 (2006).</li><li>Lou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-enhanced+adhesion+regulated+by+a+selectin+interdomain+hinge.">Flow-enhanced adhesion regulated by a selectin interdomain hinge.</a> J. Cell. Biol. 174, 1107-1117 (2006).</li><li>Yago, T., Zarnitsyna, V. I., Klopocki, A. G., McEver, R. 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=Transport+governs+flow-enhanced+cell+tethering+through+L-selectin+at+threshold+shear.">Transport governs flow-enhanced cell tethering through L-selectin at threshold shear.</a> Biophys. J. 92, 330-342 (2007).</li><li>Evans, E., Berk, D., Leung, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detachment+of+agglutinin-bonded+red+blood+cells.+I.+Forces+to+rupture+molecular-point+attachments.">Detachment of agglutinin-bonded red blood cells. I. Forces to rupture molecular-point attachments.</a> Biophys. J. 59, 838-848 (1991).</li><li>Zarnitsyna, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+in+receptor-ligand-mediated+cell+adhesion.">Memory in receptor-ligand-mediated cell adhesion.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 18037-18042 (2007).</li></ol>

Met succes gebruik van de micropipet hechting frequentie test moet men overwegen een aantal kritische stappen. Controleer eerst of de specifieke interactie van de receptor-ligand systeem van belang vast te leggen. Niet-specifieke controle metingen (zie Fig. 3, 4) zorgen voor de specificiteit. Idealiter zou niet-specifieke aanhechting waarschijnlijkheden lager zijn dan 0,05 voor alle contact tijd duur en om een ​​significant verschil tussen de specifieke en niet-specifieke aanhechting kansen voor elk tijdstip te hebb...

<table border="1"><tbody><tr><th> Naam van het reagens </th><th> Vennootschap </th><th> Catalogus # </th><th> Reacties </th></tr><tr><td> 10x PBS </td><td> BioWhittaker </td><td> 17-517Q </td><td> Verdun tot 1x met gedeïoniseerd water voor gebruik </td></tr><tr><td> Vacutainer EDTA </td><td> BD </td><td> 366643 </td><td> RBC isolatie </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 isolatie </td></tr><tr><td> Adenine </td><td> Sigma-Aldrich </td><td> A2786 </td><td> EAS-45 voorbereiding </td></tr><tr><td> D-glucose (dextrose) </td><td> Sigma-Aldrich </td><td> G7528 </td><td> EAS-45 voorbereiding </td></tr><tr><td> D-Mannitol </td><td> Sigma-Aldrich </td><td> 6360 </td><td> EAS-45 voorbereiding </td></tr><tr><td> Natriumchloride (NaCl) </td><td> Sigma-Aldrich </td><td> S7653 </td><td> EAS-45 voorbereiding </td></tr><tr><td> Natriumfosfaat, tweebasisch (Na &amp; # x2082; HPO ₄) </td><td> Fisher Scientific </td><td> S374 </td><td> EAS-45 voorbereiding </td></tr><tr><td> L-glutamine </td><td> Sigma-Aldrich </td><td> G5763 </td><td> EAS-45 voorbereiding </td></tr><tr><td> Biotine-X-NHS </td><td> Calbiochem </td><td> 203188 </td><td> RBC biotinylatie </td></tr><tr><td> Dimethylformamide (DMF) </td><td> Thermo Scientific </td><td> 20673 </td><td> RBC biotinylatie </td></tr><tr><td> Boraat buffer (0,1 M) </td><td> Electron Microscopy Sciences </td><td> 11455-90 </td><td> RBC biotinylatie </td></tr><tr><td> Streptavidine </td><td> Thermo Scientific </td><td> 21125 </td><td> Ligand functionaliseren </td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> A0336 </td><td> Ligand functionaliseren </td></tr><tr><td> Quantibrite PE Beads </td><td> BD Biosciences </td><td> 340495 </td><td> Dichtheid kwantificering </td></tr><tr><td> Flowcytometer </td><td> BD Immunocytometry Systems </td><td> BD LSR II </td><td> Dichtheid Quanidentificatie </td></tr><tr><td> Capillaire Tube 0.7-1.0mm x 30 &quot; </td><td> Kimble Glass Inc </td><td> 46485-1 </td><td> Micropipet trekken </td></tr><tr><td> Mineral Oil </td><td> Fisher Scientific </td><td> BP2629-1 </td><td> Kamer montage </td></tr><tr><td> Microscoop Dekglas </td><td> Fisher Scientific </td><td> 12-544-G </td><td> Kamer montage </td></tr><tr><td> PE α-menselijke CD11a Kloon HI 111 </td><td> eBioscience </td><td> 12-0119-71 </td><td> Reagens voor Fig.1 </td></tr><tr><td> PE anti-humaan CD54 </td><td> eBioscience </td><td> 12-0549 </td><td> Reagens voor Fig.1 </td></tr><tr><td> Muis IgG1 isotype controle PE </td><td> eBioscience </td><td> 12-4714 </td><td> Reagens voor Fig.1 </td></tr><tr><td> hydraulische micromanipulator </td><td> Narishige </td><td> MO-303 </td><td> Micropipet systeem </td></tr><tr><td> Mechanische manipulator </td><td> Newport </td><td> 461-xyz-m, SM-13, DM-13 </td><td> Micropipet systeem </td></tr><tr><td> piëzo-elektrische vertaler </td><td> Physik Instrumente </td><td> P-840 </td><td> Micropipet systeem </td></tr><tr><td> LabVIEW </td><td> National Instruments </td><td> Versie 8.6 </td><td> Micropipet systeem </td></tr><tr><td> DAQ board </td><td> National Instruments </td><td> USB-6008 </td><td> Micropipet systeem </td></tr><tr><td> Optische tafel </td><td> Kinetics Systems </td><td> 5200 Series </td><td> Micropipet systeem </td></tr></tbody></table>

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

De micropipet hechting test werd ontwikkeld in 1998 tot twee-dimensionale (2D) receptor-ligand bindingskinetiek een te meten. De test maakt gebruik van een menselijke rode bloedcellen (RBC) als hechting sensor en presenterende cel voor een van de interagerende moleculen. Er werken micromanipulatie aan de RBC in contact te brengen met een andere cel die de andere interactie molecuul tot uitdrukking met precies gecontroleerde zone en tijd in een band de vorming mogelijk te maken. De hechting gebeurtenis wordt gedetecteerd als RBC rek op te trekken de twee cellen uit elkaar. Door het controleren van de dichtheid van de liganden geïmmobiliseerd op het oppervlak van RBC, is de kans op aanhechting bewaard in mid-range tussen 0 en 1. De hechting kans is geschat op basis van de frequentie van hechting gebeurtenissen in een opeenvolging van herhaald contact cycli tussen de twee cellen voor een bepaalde tijd contact. Het variëren van de contacttijd genereert een bindend curve. Montage van een probabilistisch model voor receptor-ligand reactiekinetiek 1 tot en met de bindendecurve geeft de 2D-affiniteit en off-rate. De test is gevalideerd met behulp van interacties van Fcγ-receptoren met IgG Fc 1-6, selectines met glycoconjugaat liganden 6-9, integrines met liganden 10-13, homotypical cadherine binding 14, T-cel-receptor en coreceptor met peptide-major histocompatibility complexen 15 - 19. De methode is gebruikt om de regelgeving van 2D-kinetiek te kwantificeren door biofysische factoren, zoals het membraan microtopology 5, membraan anker 2, moleculaire oriëntatie en lengte 6, vervoerder stijfheid 9, kromming 20, en impingement werking 20, evenals biochemische factoren, zoals modulatoren van het cytoskelet en membraan micro-omgeving waar de interagerende moleculen wonen en het oppervlak organisatie van deze moleculen 15,17,19. De methode is ook gebruikt to onderzoek de gelijktijdige binding van dual-receptor-ligand soorten 3,4, en 19 trimolecular interacties met behulp van een aangepast model 21. Het grote voordeel van de methode is dat deze studie van receptoren in hun eigen omgeving membraan mogelijk maakt. De resultaten kunnen zeer verschillend zijn van de verkregen die gebruik maken van gezuiverd receptoren 17. Het staat ook studie van de receptor-ligand interacties in een sub-seconde tijdschaal met temporele resolutie ver buiten de typische biochemische methoden. Ter illustratie van de micropipet hechting frequentie methode, tonen we kinetiek meting van intercellulaire adhesie molecule 1 (ICAM-1) gefunctionaliseerde op RBC binding aan integrine α L β 2 op neutrofielen met een dimeer E-selectine in de oplossing voor α L β 2 te activeren.

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

Een hechting frequentie test voor het meten van receptor-ligand interactie kinetiek wanneer beide moleculen zijn verankerd op het oppervlak van de interagerende cellen is beschreven. Deze mechanisch-gebaseerde test wordt geïllustreerd met behulp van een micropipet-druk menselijke rode bloedcel als adhesie sensor en integrine αLβ2 en intercellulaire adhesie molecuul-1 als interactie-receptoren en liganden.

Hechting Frequency Assay voor In Situ Kinetiek Analyse van de Cross-junctionele moleculaire interacties in de cel-cel-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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Ontwikkeling van Amelogenine-chitosan Hydrogel voor<em&gt; In Vitro</em&gt; Emaille Hergroei met een dichte Interface

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

Het meten van TCR-pMHC Binding<em&gt; In Situ</em&gt; Met een FRET-gebaseerde Microscopie 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

Een Tripeptide-gestabiliseerde Nanoemulsion van oliezuur

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

Milieu dynamische mechanische analyse te voorspellen de verzachtende werking van neurale implantaten

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

Bioparticle Microarrays voor Chemotactic en Moleculaire Analyse van menselijke Neutrofiele Zwermen 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 voor seriële kristallografie metingen bij de Australische 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

Moleculaire veerconstante analyse door biomembrane force probe spectroscopie

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