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 studien ble støttet av NIH stipend R01HL091020, R01HL093723, R01AI077343, og R01GM096187.

1. RBCs isolasjon fra fullblod <ol><li> Forbered EAS-45 løsninger. Vei opp alle ingredienser fra tabell I og oppløses i 100-200ml av DI vann. Tilsett vann til å gjøre 1000ml løsning og juster pH til 8,0. Filter og delmengde av 50ml. Fryses ved -20 ° C for lagring. </li></ol> Merk: Step 1.2 skal utføres av en utdannet medisinsk faglig som for eksempel en sykepleier, med en Institutional Review Board godkjent protokollen. <ol start="2"><li> Tegn 3-5ml av blod fra media...

<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. 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>

For å kunne bruke mikropipette vedheft frekvens analysen bør man vurdere flere kritiske trinn. Først, sørg for å registrere spesifikke interaksjonsstudier for reseptor-ligand system av interesse. Uspesifikke kontroll målinger (jf. fig. 3, 4) sikre spesifisitet. Ideelt sett bør uspesifikk adhesjon sannsynligheter være under 0,05 for all kontakt tid varigheter og å ha en signifikant forskjell mellom de spesifikke og uspesifikke vedheft sannsynligheter for hvert tidspunkt. Ulike metoder kan brukes til å feste lig...

<table border="1"><tbody><tr><th> Navn på reagens </th><th> Firma </th><th> Katalog # </th><th> Kommentarer </th></tr><tr><td> 10x PBS </td><td> BioWhittaker </td><td> 17-517Q </td><td> Fortynn til 1x med avionisert vann før bruk </td></tr><tr><td> Vacutainer EDTA </td><td> BD </td><td> 366643 </td><td> RBCs isolasjon </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> RBCs isolasjon </td></tr><tr><td> Adenin </td><td> Sigma-Aldrich </td><td> A2786 </td><td> EAS-45 forberedelse </td></tr><tr><td> D-glukose (druesukker) </td><td> Sigma-Aldrich </td><td> G7528 </td><td> EAS-45 forberedelse </td></tr><tr><td> D-Mannitol </td><td> Sigma-Aldrich </td><td> 6360 </td><td> EAS-45 forberedelse </td></tr><tr><td> Natriumklorid (NaCl) </td><td> Sigma-Aldrich </td><td> S7653 </td><td> EAS-45 forberedelse </td></tr><tr><td> Natriumfosfat, Dibasic (Na &amp; # x2082; HPO ₄) </td><td> Fisher Scientific </td><td> S374 </td><td> EAS-45 forberedelse </td></tr><tr><td> L-glutamin </td><td> Sigma-Aldrich </td><td> G5763 </td><td> EAS-45 forberedelse </td></tr><tr><td> Biotin-X-NHS </td><td> Calbiochem </td><td> 203188 </td><td> RBCs biotinylation </td></tr><tr><td> Dimethylformamide (DMF) </td><td> Thermo Scientific </td><td> 20673 </td><td> RBCs biotinylation </td></tr><tr><td> Borat Buffer (0.1m) </td><td> Elektronmikroskopi Sciences </td><td> 11455-90 </td><td> RBCs biotinylation </td></tr><tr><td> Streptavidin </td><td> Thermo Scientific </td><td> 21125 </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 Perler </td><td> BD Biosciences </td><td> 340495 </td><td> Tetthet kvantifisering </td></tr><tr><td> Flowcytometer </td><td> BD Immunocytometry Systems </td><td> BD LSR II </td><td> Tetthet Quantification </td></tr><tr><td> Kapillarrør 0.7-1.0mm x 30 &quot; </td><td> Kimble Glass Inc. </td><td> 46485-1 </td><td> Mikropipette trekke </td></tr><tr><td> Mineral Oil </td><td> Fisher Scientific </td><td> BP2629-1 </td><td> Chamber montering </td></tr><tr><td> Mikroskop Cover Glass </td><td> Fisher Scientific </td><td> 12-544-G </td><td> Chamber montering </td></tr><tr><td> PE α-menneskelige CD11a Clone HI 111 </td><td> eBioscience </td><td> 12-0119-71 </td><td> Reagens for Fig.1 </td></tr><tr><td> PE anti-human CD54 </td><td> eBioscience </td><td> 12-0549 </td><td> Reagens for Fig.1 </td></tr><tr><td> Mouse IgG1 isotype Kontroll PE </td><td> eBioscience </td><td> 12-4714 </td><td> Reagens for Fig.1 </td></tr><tr><td> hydrauliske micromanipulator </td><td> Narishige </td><td> MO-303 </td><td> Mikropipette system </td></tr><tr><td> Mekanisk manipulator </td><td> Newport </td><td> 461-xyz-m, SM-13, DM-13 </td><td> Mikropipette system </td></tr><tr><td> piezoelektriske oversetter </td><td> Physik Instrumente </td><td> P-840 </td><td> Mikropipette system </td></tr><tr><td> LabVIEW </td><td> National Instruments </td><td> Versjon 8.6 </td><td> Mikropipette system </td></tr><tr><td> DAQ bord </td><td> National Instruments </td><td> USB-6008 </td><td> Mikropipette system </td></tr><tr><td> Optisk tabellen </td><td> Kinetics Systems </td><td> 5200 Series </td><td> Mikropipette system </td></tr></tbody></table>

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

Den mikropipette vedheft analysen ble utviklet i 1998 for å måle to-dimensjonale (2D) reseptor-ligand binding kinetikk 1. Analysen bruker en menneskelig røde blodlegemer (RBC) som vedheft sensor og presentere celle for en av de vekselvirkende molekyler. Det sysselsetter mikromanipulasjonsprosedyre å bringe RBC i kontakt med en annen celle som uttrykker den andre samspill molekylet med presist kontrollert område og tid for å muliggjøre bindingen formasjon. Vedheft hendelse er oppdaget som RBC forlengelse på å trekke de to cellene fra hverandre. Ved å kontrollere tettheten av ligander immobilisert på RBC overflaten, er sannsynligheten for vedheft holdt i mid-range mellom 0 og 1. Vedheft Sannsynligheten er beregnet fra hyppigheten av vedheft hendelser i en sekvens av gjentatt kontakt sykluser mellom de to cellene for en gitt kontakt tid. Varierende kontakten tiden genererer en bindende kurve. Fitting en sannsynlighets modell for reseptor-ligand reaksjonskinetikk 1 til bindendekurve returnerer 2D affinitet og off-rate. Analysen har blitt validert ved hjelp av interaksjoner av Fcγ reseptorer med IgG 1-6 Fc, selectins med glycoconjugate ligander 6-9, integriner med ligander 10-13, homotypical cadherin 14 binding, T-celle reseptor og coreceptor med peptid-dur histocompatibility komplekser 15 - 19. Metoden har vært brukt til å kvantifisere reguleringer av 2D kinetikk ved biofysiske faktorer, som membranen microtopology 5, membran anker 2, molekylær orientering og 6 lengde, carrier stivhet 9, kurvatur 20, og impingement force 20, samt biokjemiske faktorer, slik som modulatorer av cytoskjelettet og membran mikromiljøet hvor samspill molekyler bor og overflaten organiseringen av disse molekylene 15,17,19. Metoden har også blitt brukt to studere samtidig binding av dual reseptor-ligand arter 3,4, og trimolecular interaksjoner 19 ved hjelp av en modifisert modell 21. Den store fordelen med metoden er at det tillater studier av reseptorer i sitt eget membran miljø. Resultatene kan være svært forskjellig fra de som ble oppnådd ved hjelp av renset reseptorer 17. Den tillater også studier av reseptor-ligand interaksjoner i en sub-sekunders tidsskala med temporal oppløsning langt utover typiske biokjemiske metoder. For å illustrere mikropipette vedheft frekvens metoden, viser vi kinetikk måling av intercellulær adhesjonsmolekyl 1 (ICAM-1) functionalized på RBCs binding til integrin α L β 2 på nøytrofile med dimeric E-selektin i løsningen for å 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 vedheft frekvens assay for måling av reseptor-ligand interaksjoner kinetikk når begge molekyler er forankret på overflater av vekselvirkende cellene er beskrevet. Denne mekanisk-baserte analysen er eksemplifisert ved hjelp av en mikropipette-trykksatt human røde blodceller som vedheft sensor og integrin αLβ2 og intercellulær adhesjonsmolekyl-1 som samspill reseptorer og ligander.

Heft frekvens analyse for In Situ Kinetics Analyse av Cross-junctional molekylære interaksjoner på 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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Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

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Utvikling av Amelogenin-kitosan Hydrogel for<em&gt; In Vitro</em&gt; Emalje Gjengroing med en Dense 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

Måling TCR-pMHC Binding<em&gt; In Situ</em&gt; Bruker en FRET basert Mikros analysen

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 Tripeptide-stabilisert Nanoemulsion av oljesyre

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

Miljømessige dynamisk mekanisk analyse å forutsi mykgjørende virkemåten av nevrale 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

Biopartikkel Mikroarrayer for kjemotaktisk og molekylær analyse av human nøytrofil svermer 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 for Serial Crystallography Målinger på den australske 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

Molekylær vårkonstantanalyse av Biomembrane Force Probe Spectroscopy

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