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

Dit werk werd gedeeltelijk ondersteund door subsidies van de National Institutes of Health (P30 HL101302 en RO1 HL109439 naar JC) en de American Heart Association (SDG 5270005 naar JC). A. Barazia werd ondersteund door een T32HL007829 NIH training subsidie.

1. Bereiding van intravitale microscoop (Figuur 1A) <ol><li> Bereid superfusie buffer (125 mM NaCl, 4,5 mM KCl, 2,5 mM CaCl2, 1 mM MgCl2 en 17 mM NaHCO3, pH 7,4). </li><li> Zet een circulatoire waterbad temperatuur van buffer en een thermo-gecontroleerde deken te handhaven op 37 ° C. Beluchten buffer met stikstofgas (5% CO2 evenwicht met stikstof). </li><li> Zet de microscoop systeem (Sutter Lambda DG-4 hoge snelheid golflengte-wisselaar, werkstation, Olympus BX61W microsco...

<ol>
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Hier beschrijven we een gedetailleerd protocol voor real-time fluorescentie intravitale microscopie heterotypische bloedplaatjes interacties op de neutrofiel geactiveerd endotheel visualiseren tijdens vasculaire ontsteking en trombose. Voorheen soortgelijke fluorescentiemicroscopische benaderingen werden gemeld aan het moleculaire mechanisme van trombusvorming en vasculaire ontsteking te bestuderen. 8,12 Sinds de heterotypische cel-cel interactie van belang kunnen zijn voor de vaso-occlusie van de schade site...

<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

Interactie van geactiveerde bloedplaatjes en leukocyten (neutrofielen voornamelijk) op het geactiveerde endotheel bemiddelt trombose en vasculaire inflammatie. 1,2 Tijdens trombusvorming op de plaats van verwonding arteriolaire, bloedplaatjes adherent aan het geactiveerde endotheel en subendotheliale matrix eiwitten ondersteunen neutrofiel hechting walsen en 3. Omgekeerd, onder venulaire inflammatoire aandoeningen, kan neutrofielen aanhanger van de geactiveerde endotheel ondersteunen hechting en de accumulatie van circulerende bloedplaatjes. Heterotypische bloedplaatjes aggregatie neutrofiel heeft sequentiële processen die specifieke receptor-counter receptor interacties tussen cellen. 4 Het is bekend dat geactiveerde endotheelcellen adhesiemoleculen zoals von Willebrand factor los wordt geïnitieerd bloedplaatjesadhesie en accumulatie onder hoge afschuifkrachten. 5 Ook geactiveerde endotheelcellen ondersteunen neutrofiele rollen en adhesie met het uitspreken van selectines eend intercellular adhesion molecule-1 (ICAM-1), respectievelijk onder lage afschuifkrachten. 4 Platelet P-selectine neutrofielen door interactie met P-selectine glycoproteïne-1 ligand (PSGL-1), en veroorzaakt daardoor activatie van neutrofielen β2 integrines en stevig hechting tussen twee celtypes. Ondanks de vooruitgang in in vitro experimenten waarin heterotypische bloedplaatjes neutrofiel interacties bepaald in volbloed of geïsoleerde cellen, 6,7 studies die niet manipuleren oxidant stress tijdens vasculaire ziekte. In dit verslag met fluorescentie-gemerkt, specifieke antilichamen tegen een muis bloedplaatjes en neutrofielen marker beschrijven we een gedetailleerde intravitale microscopische protocol heterotypische interactie van bloedplaatjes en neutrofielen op de geactiveerde endotheel volgen tijdens TNF-α-geïnduceerde ontsteking of na laser geïnduceerde letsel bij cremaster spier haarvaten van levende muizen.

Behulp van een gedetailleerde intravitale microscopie analyse heterotypische bloedplaatjes neutrophil interacties op het geactiveerde endotheel werden gevisualiseerd door infusie van fluorescent gelabelde antilichamen tegen bloedplaatjes (CD42c) of neutrofielen marker (Gr-1) in levende muizen. In een model van TNF-α-geïnduceerde ontsteking venulaire werden de meeste rollen neutrofielen stabiel gehecht aan het endotheel vermoedelijk door interactie van geactiveerde β2 integrinen met ICAM-1...

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

Hier beschrijven we een experimentele techniek van fluorescentie intravitale microscopie heterotypische bloedplaatjes interacties op de neutrofiel geactiveerd endotheel visualiseren tijdens vasculaire inflammatie en trombose in levende muizen. Deze microscopische techniek waardevol zal zijn voor het moleculaire mechanisme van vasculaire ziekte te bestuderen en farmacologische middelen testen onder pathofysiologische omstandigheden.

Real-time beeldvorming van heterotypische Bloedplaatjes-neutrofiele Interacties op het geactiveerd endotheel tijdens vasculaire ontsteking en trombose in Live Muizen

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

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