Name: monitoring immune cells trafficking fluorescent prion rods hours after intraperitoneal infection
Uploaded: 2010-11-19T17:30:00
Description: Watch this Scientific Journal Video about Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection at JoVE.com

Wij danken Steve McBryant en Jeff Hansen voor hulp bij ultracentrifugeren en Patti Kiser voor hulp met de muis hanteren. Het Nationaal Instituut voor Neurologische Ziekten en Stroke bij de National Institutes of Health, subsidie ​​5R01NS056379-02 gefinancierde dit werk.

<html> 1. Zuiveren en Labeling Prion Rods Dit protocol is een bewerking van een eerder gepubliceerde 18 <ol><li> Homogeniseren 100 gram prion-geïnfecteerd hersenweefsel in 900 ml ijskoud homogeniseren buffer (HB, 1X PBS met 320 mM sucrose, 150 mM NaCl en 4 mm EDTA) 1 min op maximale snelheid in een commerciële blender, daarna 2 min op ijs. Herhaal dit drie keer. </li><li> Centrifuge homogenaat gedurende 10 min bij 3000 xg en 4 ° C. Verwijderen en opslaan supernatant op ijs. Re...

<ol>
	<li>Klein, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+crucial+role+for+B+cells+in+neuroinvasive+scrapie.">A crucial role for B cells in neuroinvasive scrapie.</a> Nature. 390, 687-687 (1997).</li><li>Mabbott, N. A., Farquhar, C. F., Brown, K. L., Bruce, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+immune+system+in+TSE+pathogenesis.">Involvement of the immune system in TSE pathogenesis.</a> Immunol Today. 19, 201-201 (1998).</li><li>Sigurdson, C. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+involvement+of+migratory+dendritic+cells+in+the+transfer+of+the+scrapie+agent+from+the+immune+to+peripheral+nervous+systems.">Assessing the involvement of migratory dendritic cells in the transfer of the scrapie agent from the immune to peripheral nervous systems.</a> J Neuroimmunol. 187, 114-114 (2007).</li><li>Mohan, J., Hopkins, J., Mabbott, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-derived+dendritic+cells+acquire+and+degrade+the+scrapie+agent+following+in+vitro+exposure.">Skin-derived dendritic cells acquire and degrade the scrapie agent following in vitro exposure.</a> Immunology. 116, 122-122 (2005).</li><li>Gilch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CpG+and+LPS+can+interfere+negatively+with+prion+clearance+in+macrophage+and+microglial+cells.">CpG and LPS can interfere negatively with prion clearance in macrophage and microglial cells.</a> FEBS J. 274, 5834-5834 (2007).</li><li>Safar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mass,+biochemical+composition,+and+physicochemical+behavior+of+the+infectious+form+of+the+scrapie+precursor+protein+monomer.">Molecular mass, biochemical composition, and physicochemical behavior of the infectious form of the scrapie precursor protein monomer.</a> Proc.Natl.Acad.Sci.U.S.A. 87, 6373-6373 (1990).</li><li>B&#252;eler, H. 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Hier laten we zien een protocol voor tagging en het bijhouden van prionen in vivo dat sterk vergemakkelijkt de controle vroege gebeurtenissen in de perifere prion infectie. Dit protocol zorgt voor een aan het verleden pogingen om de controle prion opname in vitro en in vivo 9 3,12 door pre-etikettering van hoog verrijkt prion inoculum op ondubbelzinnige wijze te onderscheiden van endogene PrP C. Omdat de besmettelijke prionen en PrP C delen dezelfde primaire am...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CWD-infected elk brain</td>
<td>&nbsp;</td>
<td>Private elk farm in Colorado</td>
<td>&nbsp;</td>
<td>Use any non-human prion-infected brain</td>
</tr>
<tr>
<td>Blender</td>
<td>&nbsp;</td>
<td>Oster</td>
<td>6694-015</td>
<td>Use any commercial blender</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>SS34 rotor</td>
<td>Use any centrifuge /rotor that can reach 3000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>50.2 Ti rotor</td>
<td>Use any ultracentrifuge /rotor that can reach 100,000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Bradford Reagent</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B6916</td>
<td>&nbsp;</td>
<tr>
<td>Complete mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>&nbsp;</td>
<tr>
<td>Sonicator</td>
<td>&nbsp;</td>
<td>Misonix</td>
<td>MP4000X</td>
<td>Use any horn or probe sonicator set to ~70% max power</td>
</tr>
<tr>
<td>DyLight antibody Labeling kit</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>53050</td>
<td>&nbsp;</td>
<tr>
<td>microcentrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>55430R</td>
<td>Use any refrigerated microcentrifuge that can achieve 13,000x g</td>
</tr>
<tr>
<td>centrifugal filter columns</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>Microcon YM-100</td>
<td>Use any filter or dialysis membrane with 100 Kd molecular weight cutoff</td>
</tr>
<tr>
<td>8-40 week-old FVB mice</td>
<td>&nbsp;</td>
<td>Charles River</td>
<td>207</td>
<td>Use any inbred mouse strain</td>
</tr>
<tr>
<td>1 &mu;m red fluorescent beads</td>
<td>&nbsp;</td>
<td>Phosphorex</td>
<td>2307</td>
<td>Use any fluorescent bead &le; 10 &mu;m</td>
</tr>
<tr>
<td>RPMI 1640 medium </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875-093</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>fluorescent antibodies</td>
<td>&nbsp;</td>
<td>BD pharmingen</td>
<td>Various</td>
<td>Use any fluorescent antibody appropriate for your application.</td>
</tr>
<tr>
<td>flow cytometer</td>
<td>&nbsp;</td>
<td>Dakocytomation</td>
<td>CyanADP</td>
<td>Use any flow cytometer capable of multicolor fluorescence detection</td>
</tr>		</tbody>
		</table>
		</div>

monitoring immune cells trafficking fluorescent prion rods hours after intraperitoneal infection

Aanwezigheid van een abnormale vorm een ​​host-gecodeerde prioneiwit (PrPC wordt vergeleken) die protease-resistente, pathologische en besmettelijke kenmerkt prionziekten zoals Chronic Wasting Disease (CWD) van hertachtigen en scrapie bij schapen. De Prion hypothese stelt dat deze abnormale conformeer de meeste of alle van de besmettelijke prion vormt. De rol van het immuunsysteem in het begin van de gebeurtenissen in perifere prion pathogenese is overtuigend aangetoond voor CWD en scrapie 1-3. Transgene en farmacologische studies bij muizen bleek een belangrijke rol van het complementsysteem in het behoud en het kopiëren van prionen vroeg na de infectie 4-6. In vitro en in vivo studies hebben ook prion retentie waargenomen door dendritische cellen 7-10, hoewel hun rol in de handel blijft onduidelijk 11-16. Macrofagen zijn soortgelijke wijze zijn betrokken in het begin van prion pathogenese, maar deze studies hebben zich gericht op gebeurtenissen die zich week na infectie 3,11,17. Deze eerdere studies ook last van het probleem van het onderscheid tussen endogene PrP C en besmettelijke prionen. Hier beschrijven we een semi-kwantitatieve, onpartijdige aanpak voor de beoordeling van prion opname van en de handel van de inenting site door immuuncellen er aangeworven. Geaggregeerde prion staven werden gezuiverd van geïnfecteerde hersenhomogenaat door detergent solubilisatie van niet-geaggregeerde eiwitten en ultracentrifugatie door middel van een sucrose kussen. Polyacrylamidegelelektroforese, Coomassie blauwe vlekken en Western blotting bevestigd herstel van sterk verrijkt prion staven in het ingehulde fractie. Prion staven waren fluorochroom-gelabeld dan intraperitoneaal geïnjecteerd in muizen. Twee uur later immuuncellen van peritoneale lavage vloeistof, milt en mediastinale en mesenteriale lymfeklieren werden getest op prion staaf vasthouden en cel subsets geïdentificeerd door multicolor flowcytometrie met behulp van markers voor de monocyten, neutrofielen, dendritische cellen, macrofagen en B-en T-cellen. Deze test kan voor het eerst directe controle van de immuuncellen het verwerven van en handel prionen in vivo binnen enkele uren na de besmetting. Deze test ook duidelijk onderscheidt besmettelijke, geaggregeerde prionen uit PrPC wordt vergeleken normaal tot expressie gebracht op gastheercellen, die kan moeilijk zijn en leiden tot interpretatie van gegevens problemen in andere testsystemen. Dit protocol kan worden aangepast aan andere inoculatie routes (orale, intraveneuze, subcutane en intranervous, bijvoorbeeld) en antigenen (geconjugeerd kralen, bacteriële, virale en parasitaire ziekteverwekkers en eiwitten, ei) ook.

Watch this Scientific Journal Video about Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection at JoVE.com

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Hier beschrijven we een nieuwe test voor het toezicht op prion opname en-handel door immuuncellen onmiddellijk na intraperitoneale inoculatie door het zuiveren en fluorescent etikettering geaggregeerde prion staven van geïnfecteerde hersenen materiaal dan het toezicht op hun opname en beweging van de injectieplaats en het karakteriseren van de cellen te bemiddelen deze gebeurtenissen.

Monitoring immuuncellen Trafficking TL-Prion Rods uur na intraperitoneale infectie

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

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