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 arbejde blev støttet delvist af tilskud fra National Institutes of Health (P30 HL101302 og RO1 HL109439 til JC) og American Heart Association (SDG 5.270.005 til JC). A. Barazia blev understøttet af en T32HL007829 NIH træning tilskud.

1. Fremstilling af intravital Microscope (figur 1A) <ol><li> Forbered superfusionssystem puffer (125 mM NaCI, 4,5 mM KCI, 2,5 mM CaCl2, 1 mM MgCl2, og 17 mM NaHCO3, pH 7,4). </li><li> Tænde en cirkulerende vandbad for at opretholde temperaturen af ​​puffer og thermo-styret tæppe ved 37 ° C. Beluftes puffer med nitrogengas (5% CO2 afbalanceret med nitrogen). </li><li> Tænd mikroskopet system (Sutter Lambda DG-4 high speed bølgelængde changer, arbejdsstation computer...

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Her beskriver vi en detaljeret protokol for real-time fluorescens intravital mikroskopi at visualisere heterotypiske blodplade-neutrofile interaktioner på aktiveret endotel under vaskulær inflammation og trombose. Tidligere blev lignende fluorescens mikroskopiske metoder rapporteret at undersøge den molekylære mekanisme for trombedannelse og vaskulær inflammation. 8,12 Da den heterotypisk celle-celle interaktion kunne være vigtigt for karokklusion på skadestedet, vil denne teknologi være et værdifuld...

<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

Interaktion af aktiverede blodplader og leukocytter (primært neutrofiler) på det aktiverede endotel medierer thrombose og vaskulære inflammation. 1,2 I trombedannelse på stedet for arteriolær skade, blodplader adhærente til aktiveret endotel og subendotheliale matrixproteiner understøtter neutrofil rulning og adhæsion. Tre Omvendt under venular inflammatoriske tilstande, kan neutrofiler adhærente til aktiveret endotel understøtte adhæsion og akkumulering af cirkulerende blodplader. Heterotypisk blodplade-neutrofil aggregering kræver sekventielle processer af den særlige receptor-modreceptor interaktioner mellem celler. 4. Det er kendt, at aktiverede endotelceller frigiver adhæsionsmolekyler, såsom von Willebrand-faktor, og indledte blodpladeadhæsion og akkumulering under høje forskydningsbetingelser. 5. Også, aktiverede endotelceller støtte neutrophil rullende og vedhæftning ved at udtrykke selectiner end intercellulært adhæsionsmolekyle-1 (ICAM-1), henholdsvis under betingelser med lav forskydning. fire Platelet P-selectin interagerer med neutrofiler til P-selectin glycoproteinligand-1 (PSGL-1), hvorved der induceres aktivering af neutrofile β2-integriner og faste adhæsion mellem to celletyper. På trods af de fremskridt inden for in vitro-forsøg, hvor heterotypiske blodplade-neutrofile interaktioner bestemt i fuldblod eller isolerede celler, 6,7 disse undersøgelser kan ikke manipulere oxidant stress forhold under vaskulær sygdom. I denne rapport, ved hjælp af fluorescens-mærkede, specifikke antistoffer mod et muse-blodplade-og neutrofil markør beskrives en detaljeret intravital mikroskopisk protokol til overvågning heterotypiske interaktioner mellem blodplader og neutrofiler til det aktiverede endotel under TNF-α-induceret inflammation eller efter laserinduceret skade i cremaster muskel mikrokar af levende mus.

Ved hjælp af en detaljeret intravital mikroskopi-analyse, heterotypiske blodplade-neutrophile interaktioner på det aktiverede endotel blev visualiseret ved infusion af fluorescens-mærkede antistoffer mod en blodplade (CD42c) eller neutrofil markør (Gr-1) i levende mus. I en model af TNF-α-induceret venular inflammation, var de fleste rullende neutrofiler stabilt klæbet til endotelet formodentlig ved interaktion af aktiverede β2-integriner med ICAM-1 under registreringen periode (3-4,5...

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 eksperimentel teknik til fluorescens intravital mikroskopi at visualisere heterotypiske blodplade-neutrophile interaktioner på aktiveret endotel under vaskulær inflammation og thrombedannelse i levende mus. Denne mikroskopisk teknologi vil være værdifuldt at undersøge den molekylære mekanisme af vaskulær sygdom og afprøve farmakologiske midler under patofysiologiske betingelser.

Real-time Billeddannelse af heterotypisk blodplade-neutrophile interaktioner på aktiveret endotel Under vaskulær inflammation og thrombedannelse 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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