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

Steve McBryant ve Jeff Hansen Biz fare kontrolü ile ilgili yardım için Ultrasantrifügasyon ve Patti Kiser yardım için teşekkür ederim. Ulusal Sağlık Enstitüleri Sinir Hastalıkları ve İnme Ulusal Enstitüsü, hibe 5R01NS056379-02 bu işi finanse edilmektedir.

<html> 1. Prion Çubuklar Arındırıcı ve Etiketleme Bu protokol daha önce yayınlanmış 18 uyarlanmıştır <ol><li> Ticari bir blender maksimum hızda 1 dakika, 2 dakika sonra buz üzerinde buz gibi homojenleştirici tampon 900 mL prion ile enfekte beyin dokusu 100 gram (HB, 320 mM sakkaroz, 150 mM NaCl ve 4mm EDTA içeren 1X PBS) karıştırılır. Üç kez tekrarlayın. </li><li> 3000 xg&#39;de 10 dakika ve 4 ° C Homojenat Santrifüj Çıkarın ve buz üzerinde supernata...

<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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+devoid+of+PrP+are+resistant+to+scrapie.">Mice devoid of PrP are resistant to scrapie.</a> Cell. 73, 1339-1339 (1993).</li></ol>

Burada periferik prion enfeksiyonu erken olayları büyük ölçüde takibini kolaylaştıran in vivo etiketleme ve izleme prionlar için bir protokol göstermektedir . Bu protokol, net bir şekilde endojen PrP C ayırt etmek için, ön-etiketleme zenginleştirilmiş prion inokulum, in vitro 9 ve in vivo 3,12 izleme prion alımını geçmişte girişimleri büyük ölçüde geliştirir. Çünkü bulaşıcı prionlar ve PrP C aynı birincil amino asit dizilimi...

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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

Anormal bir formu varlığı, proteaz, dayanıklı, patolojik ve bulaşıcı bir host-kodlanmış prion proteini (PrPC) Kronik Wasting koyun cervids ve scrapie hastalığı (CWD) olarak prion hastalıkları karakterize etmektedir. Prion hipotez bu anormal conformer çoğu veya tüm bulaşıcı prion teşkil ettiğini ileri sürmektedir. Periferik prion patogenezinde erken olaylar bağışıklık sisteminin rolü ikna edici CWD ve scrapie 1-3 kanıtlanmıştır. Farelerde Transgenik ve farmakolojik çalışmalar sonrası erken dönemde enfeksiyon 4-6 prionlar istinat ve kopyalayan Kompleman sisteminin önemli bir rolü ortaya koymuştur. 7-10 dendritik hücreler tarafından prion tutma gözlemledik in vitro ve in vivo çalışmalar, insan ticareti rollerini olmaya devam etmekle beraber, 11-16 belirsiz. Makrofajlar benzer erken prion patogenezinde suçlanmıştır, ancak bu çalışmalar, enfeksiyon 3,11,17 hafta sonra meydana gelen olaylar üzerine odaklanmıştır . Bu ve önceki çalışmalarda da endojen PrP C ve bulaşıcı prionlar arasındaki farklılıklarda sorun muzdarip. İşte biz orada alınan bağışıklık hücreleri tarafından aşılama sitesinden prion alımı ve ticareti değerlendirmek için bir semikantitatif, tarafsız bir yaklaşım açıklar. Toplulaştırılmış prion çubuklar olmayan toplanan proteinleri ve sakaroz yastık üzerinden Ultrasantrifügasyon deterjan çözünürleştirme ile enfekte beyin Homojenat arınmış. Poliakrilamid jel elektroforezi, Coomassie mavi boyama ve zenginleştirilmiş pelet fraksiyonu prion çubuklar batı blotting teyit kurtarma. Prion çubuklar farelere florokrom-etiketli enjekte intraperitoneal. İki saat sonra, peritoneal lavaj sıvısı, dalak ve mediastinal ve mezenterik lenf düğümleri bağışıklık hücreleri prion çubuk tutma ve monositler, nötrofil, dendritik hücreler, makrofajlar ve B ve T hücreleri için işaretleri kullanarak çok renkli akım sitometri ile tespit edilen hücre alt grupları ölçüldü. Bu testte, enfeksiyondan sonra saat içinde bağışıklık elde hücreleri ve in vivo kaçakçılığı prionlar ilk kez doğrudan izleme sağlar . Bu testte de zor ve diğer test sistemlerinde veri yorumlama sorunları neden olabilir normalde ana hücreleri üzerinde ifade PrPC bulaşıcı, toplu prion, açıkça ayırır. Bu protokol, diğer aşılama yolları (oral, intravenöz, intranervous ve derialtı, örneğin) ve aynı zamanda antijenler (konjuge boncuk, viral, bakteriyel ve paraziter patojenler ve proteinler, yumurta) adapte edilebilir.

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

Burada hemen arındırıcı intraperitoneal aşılama ve floresan sonra enjeksiyon yerinde alımı ve hareket izleme ve bu olaylara aracılık eden hücreler karakterize enfekte beyin malzeme toplanan prion çubuklar etiketleme prion alımı ve ticareti bağışıklık hücreleri tarafından izlenmesi için yeni bir tahlil açıklar.

İntraperitoneal Enfeksiyon sonra Bağışıklık Hücreleri Ticareti Floresan Prion Çubuklar Saat İzlenmesi

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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