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

Ce travail a été financé en partie par des subventions des Instituts nationaux de la santé (P30 HL101302 et RO1 HL109439 JC) et l&#39;American Heart Association (SDG 5270005 JC). A. Barazia a été soutenu par une subvention du NIH T32HL007829 formation.

1. Préparation du microscope intravitale (figure 1A) <ol><li> Préparer un tampon surfusion (125 mM NaCl, 4,5 mM KCl, 2,5 mM de CaCl 2, MgCl 2 1 mM, et 17 mM de 3 NaHCO, pH 7,4). </li><li> Activer un bain d&#39;eau pour maintenir la température de circulation de tampon et une couverture thermo-contrôlé à 37 ° C. Aérer tampon avec de l&#39;azote gazeux (5% de CO 2 en balance avec de l&#39;azote). </li><li> Allumez le système de microscope (Sutter Lambda DG-4 haute vit...

<ol>
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Ici, nous décrivons un protocole détaillé pour la microscopie en temps réel intravitale fluorescence pour visualiser hétérotypiques plaquettes neutrophiles interactions sur l&#39;endothélium activé lors de l&#39;inflammation vasculaire et la thrombose. Auparavant, similaires fluorescence approches microscopiques ont été signalés à étudier le mécanisme moléculaire de la formation de thrombus et de l&#39;inflammation vasculaire. 8,12 Depuis le hétérotypique interaction cellule-cellule pourrait ...

<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

L&#39;interaction des plaquettes activées et des leucocytes neutrophiles (essentiellement) sur l&#39;endothélium activé médie la thrombose et l&#39;inflammation vasculaire. 1,2 Au cours de la formation de thrombus au site de la lésion artériolaire, plaquettes adhérentes à l&#39;endothélium activé et protéines de la matrice de support de roulement sous-endothéliales des neutrophiles et d&#39;adhérence. 3 Inversement, dans des conditions inflammatoires veinules, les neutrophiles adhérents à l&#39;endothélium activé peut prendre en charge l&#39;adhésion et l&#39;accumulation des plaquettes circulantes. Hétérotypique plaquettes agrégation des neutrophiles nécessite des processus séquentiels par les interactions spécifiques des récepteurs à gré entre les cellules. 4 Il est connu que les cellules endothéliales activées libèrent des molécules d&#39;adhésion telles que le facteur von Willebrand, amorçant ainsi l&#39;adhésion des plaquettes et accumulation dans les conditions de cisaillement élevées. 5 En outre, cellules endothéliales activées en charge de roulement et l&#39;adhérence des neutrophiles par les sélectines exprimant uneMolécule d-1 d&#39;adhésion intercellulaire (ICAM-1), respectivement, dans des conditions de faible cisaillement. 4 plaquettes P-sélectine interagit avec les neutrophiles par la P-sélectine ligand glycoprotéine-1 (PSGL-1), ce qui induit l&#39;activation des neutrophiles de β2 intégrines et fermes l&#39;adhérence entre deux types de cellules. Malgré les progrès réalisés dans des expériences in vitro dans lequel hétérotypiques plaquettes neutrophiles interactions sont déterminées dans le sang ou des cellules isolées, 6,7 ces études ne peuvent pas manipuler les conditions de stress oxydant au cours de la maladie vasculaire. Dans ce rapport, l&#39;aide à marquage fluorescent, des anticorps spécifiques contre une souris plaquettes et des neutrophiles marqueur, nous décrivons un protocole détaillé intravitale microscopique pour surveiller les interactions hétérotypiques de plaquettes et de neutrophiles sur l&#39;endothélium activé lors de TNF-α induite par l&#39;inflammation ou à la suite induite par laser blessure musculaire crémaster microvaisseaux des souris vivantes.

En utilisant une analyse détaillée microscopie intravitale, hétérotypiques neutrophiles interactions plaquettes sur l&#39;endothélium activé ont été visualisées par perfusion d&#39;anticorps à marquage fluorescent contre une plaquette (CD42c) ou des neutrophiles marqueur (Gr-1) dans des souris vivantes. Dans un modèle de TNF-α induite par l&#39;inflammation des veinules, les neutrophiles ont été les plus roulement stable adhéré à l&#39;endothélium probablement par interacti...

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

Nous rapportons ici une technique expérimentale de la microscopie de fluorescence intravitale de visualiser hétérotypiques plaquettes neutrophiles interactions sur l&#39;endothélium activé lors de l&#39;inflammation vasculaire et la formation de thrombus chez des souris vivantes. Cette technologie microscopique seront très utiles pour étudier le mécanisme moléculaire de la maladie vasculaire et de tester des agents pharmacologiques dans des conditions physiopathologiques.

Imagerie en temps réel des hétérotypique plaquettes neutrophiles interactions sur l&#39;endothélium activé lors de l&#39;inflammation vasculaire et la formation de thrombus chez des souris vivantes

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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