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

Agradecemos a Steve McBryant e Jeff Hansen para ajudar com ultracentrifugação e Kiser Patti para ajudar no tratamento de mouse. O Instituto Nacional de Doenças Neurológicas e Derrames no National Institutes of Health, conceder 5R01NS056379-02 financiado este trabalho.

<html> 1. Purificação e Rotulagem Rods Prion Este protocolo foi adaptado de um 18 previamente publicados <ol><li> Homogeneizar 100 gramas de prion infectados tecido cerebral em 900 mL de tampão de homogeneização gelada (HB, 1X PBS contendo sacarose 320 mM, 150 mM NaCl e 4 mM EDTA) 1 min na velocidade máxima em um liquidificador comercial, em seguida, 2 min no gelo. Repita três vezes. </li><li> Centrífuga homogeneizado por 10 min a 3000 xg e 4 ° C. Retire e guarde o sobre...

<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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Aqui demonstramos um protocolo para a marcação e rastreamento de prions in vivo que muito facilita o monitoramento de eventos no início de príons periféricos. Este protocolo aumenta muito em tentativas passadas de captação de prion de monitoramento in vitro 9 e in vivo 3,12 por pré-inóculo rotulagem prion altamente enriquecido para diferenciá-lo de forma inequívoca endógena PrP C. Porque príons infecciosos e PrP C compartilhar a mesma se...

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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

Presença de uma forma anormal de uma proteína prion host-codificado (PrPC) que é resistente à protease, patológicas e infecciosas caracteriza doenças de príon, como a doença crônica Wasting (CWD) de cervídeos e scrapie em ovinos. A hipótese de Prion afirma que esta conformação anormal constitui a maior parte ou todo o príon infeccioso. O papel do sistema imunológico em eventos precoces na patogênese da prion periféricos tem sido convincentemente demonstrada por CWD e 1-3 scrapie. Transgênicos e farmacológicas estudos em ratos revelou um importante papel do sistema complemento na manutenção e replicando prions início após a infecção 4-6. In vitro e in vivo estudos também observaram retenção de prion pelas células dendríticas 10/07, apesar de seu papel no tráfico permanece claro 11-16. Macrófagos têm igualmente sido implicados na patogênese da prion cedo, mas esses estudos concentraram-se em eventos que ocorrem semanas após a infecção 3,11,17. Estes estudos anteriores também sofrem com o problema da diferenciação entre endógena PrP C e príons infecciosos. Aqui nós descrevemos uma abordagem imparcial semiquantitativo para avaliar a absorção de príon eo tráfico a partir do sítio de inoculação de células do sistema imunológico recrutados lá. Hastes prion agregados foram purificados a partir de homogenato de cérebro infectado por solubilização de detergente não agregados de proteínas e ultracentrifugação através de uma almofada de sacarose. Eletroforese em gel de poliacrilamida, coomassie coloração azul e recuperação de mata-borrão ocidental confirmados de varas prion altamente enriquecido na fração peletizada. Hastes príon foram fluorocromo marcado por via intraperitoneal, em seguida, injetadas em ratos. Duas horas mais tarde células imunológicas de lavado peritoneal, baço e gânglios linfáticos do mediastino e mesentéricos foram ensaiadas para a retenção de prion haste e subpopulações de células identificadas por multicolor citometria de fluxo utilizando marcadores para monócitos, neutrófilos, células dendríticas, macrófagos e células B e T. Este ensaio permite o monitoramento direto da primeira vez que células do sistema imunológico aquisição e prions tráfico in vivo dentro de horas após a infecção. Este ensaio também diferencia claramente infecciosas, prions agregadas de PrPC normalmente expressa em células do hospedeiro, que pode ser difícil e levar a problemas de interpretação de dados em sistemas de ensaio de outros. Este protocolo pode ser adaptado para outras rotas de inoculação (oral, intravenosa, subcutânea e intranervous, por exemplo) e antígenos (grânulos conjugados, patógenos bacterianos, virais e parasitárias e proteínas, o ovo) também.

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

Aqui nós descrevemos um ensaio de romance para o monitoramento e captação de prion tráfico por células do sistema imunológico imediatamente após a inoculação intraperitoneal de purificação e fluorescente rotulagem varas prion agregados de material cerebral infectado acompanhamento da sua absorção e movimento a partir do local da injeção e caracterizar as células mediadoras desses eventos.

Monitoramento células imunes Tráfico Rods Horas Prion fluorescente após a infecção intraperitoneal

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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