Name: real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice
Uploaded: 2013-04-02T13:00:00
Description: Watch this Scientific Journal Video about Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mic

Dette arbeidet ble støttet delvis med tilskudd fra National Institutes of Health (P30 HL101302 og RO1 HL109439 til JC) og American Heart Association (SDG 5270005 til JC). A. Barazia ble støttet av en T32HL007829 NIH trening stipend.

1. Utarbeidelse av Intravital mikroskop (figur 1A) <ol><li> Forbered superfusjons buffer (125 mM NaCl, 4,5 mM KCl, 2,5 mM CaCl 2, 1 mM MgCl 2, og 17 mM 3 NaHCO, pH 7,4). </li><li> Slå på en sirkulasjons vannbad for å opprettholde temperaturen av buffer og et termo-kontrollert teppe ved 37 ° C. Aerate buffer med nitrogengass (5% CO 2 balanseres med nitrogen). </li><li> Slå på mikroskopet systemet (Sutter Lambda DG-4 høy hastighet bølgelengde veksler, arbeidsstasjon da...

<ol>
	<li>Wagner, D. D., Frenette, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vessel+wall+and+its+interactions.">The vessel wall and its interactions.</a> Blood. 111, 5271-5281 (2008).</li><li>Nieswandt, B., Kleinschnitz, C., Stoll, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischaemic+stroke:+a+thrombo-inflammatory+disease.">Ischaemic stroke: a thrombo-inflammatory disease.</a> J. Physiol. 589, 4115-4123 (2011).</li><li>Yang, J., Furie, B. C., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+P-selectin+glycoprotein+ligand-1:+its+role+as+a+selectin+counterreceptor+in+leukocyte-endothelial+and+leukocyte-platelet+interaction.">The biology of P-selectin glycoprotein ligand-1: its role as a selectin counterreceptor in leukocyte-endothelial and leukocyte-platelet interaction.</a> Thromb. Haemost. 81, 1-7 (1999).</li><li>Zarbock, A., Polanowska-Grabowska, R. K., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-neutrophil-interactions:+linking+hemostasis+and+inflammation.">Platelet-neutrophil-interactions: linking hemostasis and inflammation.</a> Blood Rev. 21, 99-111 (2007).</li><li>Chen, J., Lopez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+platelets+with+subendothelium+and+endothelium.">Interactions of platelets with subendothelium and endothelium.</a> Microcirculation. 12, 235-246 (2005).</li><li>Konstantopoulos, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venous+levels+of+shear+support+neutrophil-platelet+adhesion+and+neutrophil+aggregation+in+blood+via+P-selectin+and+beta2-integrin.">Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin.</a> Circulation. 98, 873-882 (1998).</li><li>Maugeri, N., de Gaetano, G., Barbanti, M., Donati, M. B., Cerletti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+platelet-polymorphonuclear+leukocyte+interactions:+new+clues+to+the+antithrombotic+properties+of+parnaparin,+a+low+molecular+weight+heparin.">Prevention of platelet-polymorphonuclear leukocyte interactions: new clues to the antithrombotic properties of parnaparin, a low molecular weight heparin.</a> Haematologica. 90, 833-839 (2005).</li><li>Hidalgo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotypic+interactions+enabled+by+polarized+neutrophil+microdomains+mediate+thromboinflammatory+injury.">Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.</a> Nat. Med. 15, 384-391 (2009).</li><li>Cho, J., Furie, B. C., Coughlin, S. R., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+extracellular+protein+disulfide+isomerase+during+thrombus+formation+in+mice.">A critical role for extracellular protein disulfide isomerase during thrombus formation in mice.</a> J. Clin. Invest. 118, 1123-1131 (2008).</li><li>Cho, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+disulfide+isomerase+capture+during+thrombus+formation+in+vivo+depends+on+the+presence+of+beta3+integrins.">Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of beta3 integrins.</a> Blood. 120, 647-655 (2012).</li><li>Gross, P. L., Furie, B. C., Merrill-Skoloff, G., Chou, J., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-versus+microparticle-mediated+tissue+factor+transfer+during+arteriolar+thrombus+development.">Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development.</a> Journal of Leukocyte Biology. 78, 1318-1326 (2005).</li><li>Falati, S., Gross, P., Merrill-Skoloff, G., Furie, B. C., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+imaging+of+platelets,+tissue+factor+and+fibrin+during+arterial+thrombus+formation+in+the+mouse.">Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.</a> Nat. Med. 8, 1175-1181 (2002).</li><li>Barthel, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha+1,3+fucosyltransferases+are+master+regulators+of+prostate+cancer+cell+trafficking.">Alpha 1,3 fucosyltransferases are master regulators of prostate cancer cell trafficking.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 19491-19496 (2009).</li><li>Trzpis, M., McLaughlin, P. M., de Leij, L. M., Harmsen, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+adhesion+molecule:+more+than+a+carcinoma+marker+and+adhesion+molecule.">Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule.</a> The American Journal of Pathology. 171, 386-395 (2007).</li><li>Junt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317, 1767-1770 (2007).</li><li>Egan, C. E., Sukhumavasi, W., Bierly, A. L., Denkers, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+multiple+functions+of+Gr-1(+)+cell+subpopulations+during+microbial+infection.">Understanding the multiple functions of Gr-1(+) cell subpopulations during microbial infection.</a> Immunologic Research. 40, 35-48 (2008).</li></ol>

Her beskriver vi en detaljert protokoll for real-time fluorescens intravital mikroskop for å visualisere heterotypic blodplatelignende nøytrofile interaksjoner på aktivert endotel under vaskulær inflammasjon og trombose. Tidligere ble lignende fluorescens mikroskopiske tilnærminger rapportert å studere molekylære mekanismen for trombedannelse og vaskulær inflammasjon. 8,12 Siden heterotypic celle-celle interaksjon kan være viktig for vaso-okklusjon på skadestedet, vil denne teknologien være et verd...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>144-55-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.9% NaCl Saline</td>
			<td>Hospira</td>
			<td>0409-4888-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira</td>
			<td>0409-2051-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 90)</td>
			<td>BD Diagnostics</td>
			<td>427421</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 10)</td>
			<td>BD Diagnostics</td>
			<td>427401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Murine TNF-&alpha;</td>
			<td>R&amp;D Systems</td>
			<td>410-MT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dylight 488- labeled rat anti-mouse CD42b antibody</td>
			<td>Emfret Analytics</td>
			<td>X488</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody</td>
			<td>BioLegend</td>
			<td>108418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NESLAB EX water bath/circulator</td>
			<td>Thermo-Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus BX61W microscope</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TH4-100 Power</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lambda DG-4</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MPC-200 multi-manipulator</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ROE-200 stage controller</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C9300 high-speed camera</td>
			<td>Hamamatsu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intensifier</td>
			<td>Video Scope International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ablation Laser</td>
			<td>Photonic Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlideBook 5.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice

Interaksjon av aktiverte blodplater og leukocytter (hovedsakelig nøytrofiler) på den aktiverte endotel medierer trombose og vaskulær inflammasjon. 1,2 Under trombedannelse på stedet av arteriolar skade, blodplater adherente til den aktiverte endotel og subendothelial matriksproteiner støtter nøytrofil rullende og heft. 3 omvendt, under venular inflammatoriske tilstander, kan neutrofiler vedheftende til aktivert endotel støtte adhesjon og akkumulering av sirkulerende blodplater. Heterotypic platelignende nøytrofil aggregering trenger sekvensielle prosesser av de spesifikke reseptor-counter reseptor interaksjoner mellom celler. 4 Det er kjent at aktivert endotelcellene frigi adhesjonsmolekyler som von Willebrand faktor, og dermed initiere blodplate adhesjon og akkumulering under høye skjærbetingelser. 5 Også, aktivert endotelceller støtte nøytrofile rullende og heft ved å uttrykke selectins end intercellulære adhesjonsmolekyl-1 (ICAM-1), henholdsvis, under lave skjærbetingelser. 4 Platelet P-selektin samhandler med nøytrofiler gjennom P-selektin glykoprotein ligand-1 (PSGL-1), for derved å indusere aktivering av nøytrofile β2 integriner og fast adhesjon mellom to celletyper. Til tross for fremskritt i in vitro eksperimenter der heterotypic blodplatelignende nøytrofile interaksjoner bestemmes i fullblod eller isolerte celler, 6,7 disse studiene ikke kan manipulere oxidant stress forhold under vaskulær sykdom. I denne rapporten bruker fluorescently-merket, spesifikke antistoffer mot en mus blodplater og nøytrofile markør, beskriver vi en detaljert intravital mikroskopisk protokoll for å overvåke heterotypic interaksjoner av blodplater og nøytrofile på den aktiverte endotel under TNF-α-indusert inflammasjon eller etter laser-indusert skade i cremaster muskel microvessels av levende mus.

Ved hjelp av en detaljert intravital mikroskopi analyse, var heterotypic blodplatelignende nøytrofile interaksjoner på aktiverte endothelium visualiseres ved infusjon av fluorescently-merkede antistoffer mot en blodplater (CD42c) eller nøytrofile markør (Gr-1) i levende mus. I en modell av TNF-α-indusert venular inflammasjon, ble de fleste rullende nøytrofile stabilt festet til endotelet formodentlig ved interaksjon av aktiverte β2 integriner med ICAM-1 under opptaket perioden (3 til ...

Watch this Scientific Journal Video about Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mic

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Her rapporterer vi en eksperimentell teknikk av fluorescens intravital mikroskop for å visualisere heterotypic blodplatelignende nøytrofile interaksjoner på aktivert endotel under vaskulær inflammasjon og trombedannelse i levende mus. Denne mikroskopiske teknologien vil være verdifullt å studere molekylære mekanismen for vaskulær sykdom og å teste farmakologiske stoffer i henhold patofysiologiske forhold.

Real-time Imaging av Heterotypic Blodplate-nøytrofile interaksjoner på Aktivert Endotelet Under vaskulær inflammasjon og trombedannelse i levende mus

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

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