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

Questo lavoro è stato sostenuto in parte da sovvenzioni dal National Institutes of Health (P30 HL101302 e RO1 HL109439 a JC) e dell&#39;American Heart Association (SDG 5.270.005 a JC). A. Barazia è stato sostenuto da una borsa di formazione T32HL007829 NIH.

1. Preparazione di Microscopio intravitale (Figura 1A) <ol><li> Preparare tampone superfusione (125 mM NaCl, 4,5 mM KCl, 2,5 mM CaCl 2, 1 mM MgCl 2, e 17 mM NaHCO 3, pH 7,4). </li><li> Attivare un bagnomaria circolatorio per mantenere la temperatura del tampone e termo-controllata coperta a 37 ° C. Aerare tampone con gas di azoto (5% di CO2 equilibrato con azoto). </li><li> Accendere il sistema di microscopio (Sutter Lambda DG-4 ad alta velocità di lunghezza d&#39;onda cam...

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Qui si descrive un protocollo dettagliato per tempo reale microscopia a fluorescenza intravitale di visualizzare eterotipiche piastrine-neutrofili interazioni sull&#39;endotelio vascolare attivato durante l&#39;infiammazione e trombosi. In precedenza, simili approcci fluorescenza microscopiche sono stati segnalati per studiare il meccanismo molecolare della formazione di trombi e infiammazione vascolare. 8,12 Poiché il heterotypic interazione cellula-cellula potrebbe essere importante per vaso-occlusione al ...

<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

Interazione delle piastrine attivate e leucociti neutrofili (principalmente) sulla trombosi attivato media endotelio e infiammazione vascolare. 1,2 Durante la formazione del trombo nel sito di lesione arteriolare, piastrine aderenti all&#39;endotelio attivato e proteine ​​della matrice subendoteliali supportare rotolamento dei neutrofili e adesione. 3 Viceversa, in venulare condizioni infiammatorie, aderenti neutrofili all&#39;endotelio attivato può sostenere l&#39;adesione e l&#39;accumulo di piastrine circolanti. Eterotipica piastrine-neutrofili aggregazione richiede processi sequenziali dagli specifici recettori-counter interazioni tra cellule recettore. 4 È noto che cellule endoteliali attivate rilasciare molecole di adesione come fattore di von Willebrand, avviando adesione piastrinica e accumulo condizioni di alto shear. 5 Inoltre, cellule endoteliali attivate supportare rotolamento dei neutrofili e adesione da selectine esprimono unmolecola-1 di adesione intercellulare d (ICAM-1), rispettivamente, in condizioni di basso attrito. 4 piastrine P-selectina interagisce con neutrofili attraverso P-selectina glicoproteina ligando-1 (PSGL-1), inducendo così l&#39;attivazione dei neutrofili β2 integrine e azienda adesione tra due tipi di cellule. Nonostante i progressi in esperimenti in vitro in cui sono determinati eterotipiche piastrine-neutrofili interazioni in sangue intero o cellule isolate, 6,7 quegli studi non può manipolare durante condizioni di stress ossidativo malattia vascolare. In questo rapporto, con marcati in fluorescenza, anticorpi specifici contro un mouse piastrinica e dei neutrofili marcatore, si descrive un dettagliato protocollo intravitale microscopica per monitorare le interazioni eterotipiche di piastrine e dei neutrofili sul endotelio attivato durante il TNF-α indotta da infiammazione o dopo laser-indotta lesioni in Cremaster microvasi muscolari di topi vivi.

Con un&#39;analisi dettagliata microscopia intravitale, eterotipiche piastrine-neutrofili interazioni sul endotelio attivato sono state visualizzate mediante infusione di anticorpi marcati con fluorescenza contro una piastrina (CD42c) o neutrofili marcatore (Gr-1) in topi vivi. In un modello di TNF-α indotta infiammazione venulare, neutrofili più laminazione sono stabilmente aderito all&#39;endotelio presumibilmente dall&#39;interazione attivati ​​β2 integrine con ICAM-1 durante il pe...

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 (Video) | JoVE

Qui riportiamo una tecnica sperimentale di fluorescenza microscopia intravitale di visualizzare eterotipiche piastrine-neutrofili interazioni sul endotelio attivato durante l&#39;infiammazione vascolare e la formazione di trombi nei topi vivi. Questa tecnologia microscopica sarà prezioso per studiare il meccanismo molecolare della malattia vascolare e per testare agenti farmacologici in condizioni fisiopatologiche.

In tempo reale di immagine degli eterotipica piastrine-neutrofili interazioni sull&#39;endotelio Attivato durante l&#39;infiammazione vascolare e la formazione di trombi in topi vivi

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

Research

JoVE Journal

Immunology and Infection

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

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

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In tempo reale immagini di leucotriene B 4 Migrazione cellulare mediata e BLT1 Interazioni con β-arrestina

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

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