Ringraziamo McBryant Steve e Jeff Hansen aiuto per ultracentrifugazione e Patti Kiser aiuto per la gestione del mouse. L&#39;Istituto Nazionale di Malattie neurologici e colpo al National Institutes of Health, concedere 5R01NS056379-02 finanziato questo lavoro.

<html> 1. Purificante e etichettatura Rods prioni Questo protocollo è adattato da quello precedentemente pubblicato 18 <ol><li> Omogeneizzare 100 grammi di prioni infetti tessuto cerebrale in 900 ml di buffer di ghiaccio freddo omogeneizzazione (HB, 1X PBS contenente 320 mM saccarosio, 150 mM NaCl e 4mm EDTA) 1 min alla massima velocità in una pubblicità frullatore, poi 2 minuti sul ghiaccio. Ripetete tre volte. </li><li> Centrifuga omogenato per 10 min a 3000 xg e 4 ° C. Rimuovere il surnatante e sul ghiaccio. Risospendere pellet in 1L HB e ripetere i punti 1.1 e 1.2. </li><li> Surnatanti piscina e centrifugare per 60 minuti a 100.000 xg, 0 ° C. Gettare il surnatante. </li><li> Risospendere pellet in 100 ml di soluzione salina tamponata Tris 1X (10 mM Tris-HCl, 0,1 mM NaCl, 1 mM EDTA) contenente 2% Triton X-100 (TBST). Stima concentrazione di proteine ​​da Bradford o test simili e regolare a 5 mg / ml in TBST. Freddo in ghiaccio per 30 min. </li><li> Centrifugare per 30 min a 100.000 xg, 0 ° C. Gettare il surnatante e lavare pellet con 50 mL TBST poi 100 mL TBS. Risospendere pellet in 100 ml di PBS 1X contenente l&#39;1% sarcosyl e cocktail inibitore della proteasi e mescolate per 120 minuti a 37 ° C. </li><li> Strato del campione su 900 ml di 320 mM saccarosio e centrifugare per 60 minuti a 100.000 xg, 10 ° C. Gettare il surnatante e risospendere il pellet in 10 ml 2.3M NaCl, 5% sarcosyl. </li><li> Sonicare campione di 5 x 40 secondi alla potenza massima del 70% a intervalli di 1 min sul ghiaccio. </li><li> Aliquota 1 ml di campione in provette Eppendorf e centrifugare per quindici minuti a 13.000 xg, 4 ° C. Lavare pellet due volte con TBS e conservare @ -70 ° C o coniugato al fluorocromo al punto 1.9. </li><li> Coniugato 1 tubo di purificato aste dei prioni con 1 fiala di DyLight 649 fluorocromo secondo il protocollo del produttore. </li><li> Scambio DyLight tampone di etichettatura per PBS da dialisi o centrifugazione attraverso le colonne del filtro. Conservare in 20 microlitri aliquote a -70 ° C al riparo dalla luce fino al momento dell&#39;uso. </li></ol> 2. Prioni inoculazione e recupero cellulare Questa sezione comprende l&#39;inoculazione intraperitoneale prioni e il recupero di cellule dal peritoneo, linfonodi mediastinici e mesenterici e la milza. <ol><li> Diluire aste prioni fluorescenti 1:50 in PBS immediatamente prima dell&#39;uso. Topo collottola dal collo di pelliccia dorsale con pollice e indice, delicatamente girare mouse per affrontare voi e frenare la coda con il dito mignolo. Supinazione del mouse, elevando torso posteriori sopra la testa e tenendolo un po &#39;a testa in giù. </li><li> Utilizzando una siringa per insulina 30G, iniettare 100 ml di aste prioni fluorescente 1 cm lateralmente alla linea mediana e 1-2 cm anteriormente alla sacroiliaca. Inserire l&#39;ago 1 centimetro attraverso la pelle rivolta a 45 ° medialmente. L&#39;uso diluizione 1:50 di perline fluorescenti o PBS solo come controlli. Incubare i topi per il tempo assegnato prima dell&#39;analisi. </li><li> Euthanize topi secondo la cura degli animali approvato ed utilizzano protocolli commissione. Iniettare 10 ml di PBS nella cavità peritoneale con una siringa da 12 ml e ago 25G. Scuotere delicatamente carcassa longitudinalmente per un minuto. Recuperare sospensione cellulare dal peritoneo utilizzando la stessa siringa e un ago 16G in un tubo da 15 ml e riserva sul ghiaccio mentre dissezione della carcassa continua. </li><li> Bagnare la parte ventrale della carcassa accuratamente con etanolo al 70%. Sollevare la pelle sulla linea mediana appena sotto la gabbia toracica con una pinza e tagliate una piccola incisione attraverso la pelle con le forbici. A partire lì, fare una incisione lungo 3-5 centimetri tra la pelle in entrambe le direzioni a testa e la coda, poi due incisioni di 2 cm dalla linea mediana lateralmente da ciascuna estremità. Staccare la spina e pelle per rivelare la cavità peritoneale e toracica. Con attenzione tagliare le sottili membrane traslucide che circondano il peritoneo e riflettono il torace per rivelare il mediastino. </li><li> Individuare e rimuovere 2-4 linfonodi mediastinici, leggermente dorsale e lateralmente adiacente al timo, medialmente adiacente all&#39;arteria brachiocefalica, vicino al lato ventrale della trachea. Individuare e rimuovere i nodi fino a sei linfonodi mesenterici, incorporati nel tessuto molle mesenterica bianco che ancora l&#39;intestino alla parete addominale posteriore. Individuare e rimuovere la milza, un lungo organo rosso scuro situato laterale della linea mediana e dorsale per l&#39;intestino nella parte sinistra dell&#39;addome. Linfonodi riserva in 1 ml di RPMI 1640 medium in ghiaccio fino dissezione è completa. </li><li> Macerare e premere linfonodi attraverso un filtro di 40 micron in 3 mL di RPMI in una capsula di Petri di plastica con lo stantuffo di una siringa da 1 ml. Sciacquare il filtro con un ulteriore 2 ml di RPMI e trasferire i 5 ml di sospensione cellulare in una provetta da 15 ml. Sciacquare la piastra Petri con altri 5 ml di RPMI e trasferimento al tubo stesso. Centrifugare 5 min a 250 xg, scartare il surnatante, risospendere il pellet cellulare in 1 ml FACS tampone (PBS 1X, 1% di albumina sierica bovina e 10 mM EDTA) e trasferire in una provetta da 1,5 ml Eppendorf. </li></ol><html> ove_title &quot;&gt; 3. colorazione della cellula e Citometria a Flusso Il seguente è un protocollo di colorazione tipica per identificare sottogruppi di cellule immunitarie utilizzando anticorpi fluorescenti contro marcatori cellulari ben caratterizzati immunitario di superficie. Tutti gli anticorpi sono stati diluiti in tampone FACS immediatamente prima dell&#39;uso. <ol><li> Contare le celle e aliquota 10 6 cellule in provette Eppendorf. Cellule centrifuga (vedi 2.7) e lavare pellet 2X con 1 mL di tampone di FACS. </li><li> Incubare cellule di 20 minuti sul ghiaccio in 100 l di 2 ng / mL ratto anti-topo CD16/32 di bloccare i recettori Fc endogena. Pellet e le cellule lavare con tampone FACS. </li><li> Aggiungere 100 ml di 1NG / mL appropriati anticorpi fluorescenti (y) IES ad ogni campione e incubare in ghiaccio per un&#39;ora. Aggiungere 100 ml di buffer di FACS solo alle cellule di controllo. Pellet e le cellule lavare due volte con tampone FACS. </li><li> Risospendere le cellule in 1 mL di tampone di ACK (150 mm NH 4 Cl, 1 mM KHCO 3 e 0.1 mM EDTA) Incubare in ghiaccio per 1 min per lisare i globuli rossi. Pellet e le cellule lavare con tampone FACS. Risospendere le cellule in 1 mL di tampone di FACS. </li><li> Raccogliere i dati dalle cellule utilizzando un citometro a flusso multicolore capace di rilevazione e di analisi, tipicamente con laser 405 nm, 488 e 633 eccitazione e 09:55 rilevatori di emissioni di fluorescenza. </li></ol> 4. Rappresentante dei risultati: Siamo purificati da prioni aste greggio, prioni infetti omogenato cerebrale (figura 1) solubilizzazione Detergente e ultracentrifugazione del alce E2 cervello omogenato (corsie 1 e 2) notevolmente arricchito per aste dei prioni (confrontare corsie 1 e 3), che è stato anche proteasi K resistente (corsia 4). È interessante notare che non digerito aste dei prioni purificata mostrato un profilo di glycoform sorprendentemente simile a omogenato E2 digerito cervello, indicando arricchimento drammatico per i prioni. Identicamente trattati con omogenato di cervello normale non ha prodotto PK-resistente PrP C (corsie 5 e 6). Citometria a flusso di popolazioni di cellule specifiche dimostrare che le cellule presentanti l&#39;antigene casa al peritoneo di due ore e conservare perline fluorescenti e bacchette prioni, come evidenziato dalla fluorescenza drammaticamente aumentata durante i controlli PBS iniettato (Figura 2). Nessun cellule raccolte dal peritoneo di PBS trattati con comandi visualizzati sopra fluorescenza di fondo (Fig. 2A-G), mentre le cellule da cordone (Fig. 2H-N) e prioni (Fig. 2O-U)-inoculato nei topi visualizzata fluorescenza significativo, soprattutto sui monociti (Fig. 2I e P), le cellule dendritiche (Fig. 2K e R) e macrofagi (Fig. 2L e S), e alcuni neutrofili (Figura 2J e Q) e un minor numero di cellule B (2T Figura). Perline e prioni portante monociti, le cellule dendritiche ei macrofagi sono stati trovati anche nei linfonodi mediastinici due ore dopo l&#39;inoculazione (Fig. 2AD e AK, AF e AM e AN e AG, rispettivamente), mentre poche o nessuna PrP CWD-cuscinetto neutrofili , le cellule B o T sono stati trovati lì (fig. 2AE e AL, AH e AI e AG e AP, rispettivamente). Abbiamo rilevato nessun perline o aste dei prioni nella milza e nei linfonodi mesenterici (dati non riportati). In toto, questi dati dimostrano che le cellule immunitarie traffico prioni in modo molto simile agli antigeni altre particelle. <table border="1"><tbody><tr><td> REAGENTE </td><td> Fluorocromo </td><td> ECCITAZIONE LASER (nm) </td><td> Picco di emissione (nm) </td></tr><tr><td> Prioni aste </td><td> DyLight 649 </td><td> 633 </td><td> 674 </td></tr><tr><td> 1 perline micron </td><td> AlexaFluor 660 </td><td> 633 </td><td> 685 </td></tr><tr><td> Anticorpi </td><td></td><td></td><td></td></tr><tr><td> CD11b </td><td> eFluor 450 </td><td> 405 </td><td> 450 </td></tr><tr><td></td><td> Ficoeritrina-Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> CD11c </td><td> R-ficoeritrina </td><td> 488 </td><td> 575 </td></tr><tr><td></td><td> Ficoeritrina-Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> Ly6C </td><td> Fluoroisothiocyante </td><td> 488 </td><td> 518 </td></tr><tr><td> Ly6G </td><td> R-ficoeritrina </td><td> 488 </td><td> 575 </td></tr><tr><td> CD21 </td><td> DyLight 488 </td><td> 488 </td><td> 518 </td></tr><tr><td> B220 </td><td> Allophycocyanin-Cy7 </td><td> 633 </td><td> 785 </td></tr><tr><td> CD3 </td><td> R-ficoeritrina </td><td> 488 </td><td> 575 </td></tr></tbody></table>&quot;&gt; Tabella 1. Proprietà spettrali di reagenti fluorescenti <img src="/files/ftp_upload/2349/2349fig1.jpg" alt="Figura 1" /> Figura 1. Purificazione di canne da prioni omogenato cerebrale infetto. Abbiamo usato un 10% omogenati cerebrali greggio da un CWD infetti cervo (E2, corsie 1 e 2) come materiale di partenza da cui ci purificati aste prioni aggregati (corsie 3 e 4). Entrambi i materiali grezzi e purificati hanno mostrato resistenza caratteristica a proteasi K trattamento (corsie 1 - 4), mentre omogenato cerebrale normale non (corsie 5 e 6). Marcatori peso molecolare sono riportate nella Kd a sinistra della macchia. BH, omogenato cerebrale. <img src="/files/ftp_upload/2349/2349fig2.jpg" alt="Figura 2" /> Figura 2. Analisi di citometria a flusso di traffico di cellule immunitarie prioni dal peritoneo ai linfonodi mediastinici due ore dopo l&#39;inoculazione. PBS (pannelli AG e V-AB), perline fluorescenti (HN e AC-AI) o di bacchette da prioni (OU, e AJ-AP) sono stati iniettate nel peritoneo di topi e le cellule raccolte dal liquido di lavaggio peritoneale (AU) o linfonodi mediastinici (V-AP), due ore dopo. Grafici nel prime cellule mostrano colonna da topi trattati con PBS (pannelli A e V), perline fluorescenti (pannello H e AC) e aste dei prioni (pannelli O e AJ). Cellule fluorescenti (punti rossi o verdi) e le cellule totale (punti grigi) vengono riportati per mostrare dimensione relativa (scatter in avanti), granularità (scatter lato) e la proporzione del totale delle cellule vive che fluorescenza. Un significativo numero di cellule caratteristico dei granulociti mantenuto perline fluorescenti e bacchette. Le cellule sono state anche colorate con anticorpi contro marcatori immunitari superficie cellulare e gated per LYG-Ly6C + monociti (pannelli B, I, P, W, AD e AK), Ly6c-CD11c-CD11b + Ly6G + neutrofili (C, J, Q, X, AE e AL), Ly6G-Ly6C-CD11b + CD11c + cellule dendritiche (D, K, R, Y, AF e AM), Ly6G-Ly6C-CD11c-CD11b + macrofagi, (E, L, S, Z, AG e AN), CD3 -B220 + CD21 + cellule B (F, M, T, AA, AH e AO) e CD21-B220-cellule T CD3 + (G, N, U, AB, AI e AP). Monociti, le cellule dendritiche ei macrofagi mantenuto perline fluorescenti e aste dei prioni nella cavità peritoneale (pannelli di I e P, K e R e L e S, rispettivamente) e trasportati a linfonodi mediastinici (pannelli dC e AK, AF e AM e AG e AN, rispettivamente), dimostrando l&#39;assorbimento simili e il traffico di queste due particelle. Neutrofili mantenuto una notevole quantità di perle (J) e un minor numero di aste (Q), ma non è riuscito a consegnarli ai linfonodi (AE e AL). Cellule B conservati e trasportati alcune aste (T e AO), ma praticamente nessun perle (M e AH). Le cellule T di traffico né perle né barre (N, U, AI e AP).

<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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P., MacPherson, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+oral+transmission+of+prion+diseases.">Dendritic cells and oral transmission of prion diseases.</a> Adv Drug Deliv Rev. 56, 901-901 (2004).</li><li>Ano, Y., Sakudo, A., Nakayama, H., Onodera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+dynamics+of+infectious+prion+protein+in+the+intestine.">Uptake and dynamics of infectious prion protein in the intestine.</a> Protein Pept Lett. 16, 247-247 (2009).</li><li>Aucouturier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infected+splenic+dendritic+cells+are+sufficient+for+prion+transmission+to+the+CNS+in+mouse+scrapie.">Infected splenic dendritic cells are sufficient for prion transmission to the CNS in mouse scrapie.</a> J Clin Invest. 108, 703-703 (2001).</li><li>Huang, F. 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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>

Nessun conflitto di interessi dichiarati.

Qui mostriamo un protocollo per il tagging e prioni monitoraggio in vivo che facilita notevolmente il monitoraggio eventi precoci nelle infezioni da prioni periferici. Questo protocollo migliora significativamente i tentativi passati a monitorare l&#39;assorbimento del prione in vitro 9 e in vivo da 3,12 pre-etichettatura inoculo prione altamente arricchito per inequivocabilmente lo differenziano da PrP C endogena. Poiché i prioni infettivi e PrP C ...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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

Presenza di una forma anomala di un host codifica della proteina prionica (PrPc) che è resistente alle proteasi, patologica e contagiosa che caratterizza le malattie da prioni come la sindrome del dimagrimento cronico (CWD) di cervidi e la scrapie nelle pecore. L&#39;ipotesi da prioni afferma che questo conformero anomala costituisce la maggior parte o tutti i prioni infettivi. Il ruolo del sistema immunitario in eventi precoci nella patogenesi dei prioni periferici è stata dimostrata in modo convincente per la malattia del dimagrimento cronico e scrapie 1-3. Transgeniche e studi farmacologici nei topi hanno rivelato un ruolo importante del sistema del complemento a mantenere e replicare i prioni presto dopo l&#39;infezione 4-6. In vitro e in studi in vivo hanno anche osservato la conservazione dei prioni da parte delle cellule dendritiche 7-10, anche se il loro ruolo nel traffico resta chiaro 11-16. I macrofagi sono similmente stati implicati nella patogenesi prioni presto, ma questi studi si sono concentrati sugli eventi che si verificano settimane dopo l&#39;infezione 3,11,17. Questi studi precedenti inoltre hanno il problema di distinguere tra endogena PrP C e prioni infettivi. Qui descriviamo una semiquantitativo, approccio imparziale per valutare l&#39;assorbimento del prione e il traffico dal sito di inoculazione da parte delle cellule immunitarie reclutate lì. Aste dei prioni aggregati sono stati purificati da omogenato cerebrale infetto da solubilizzazione di detergente non aggregata proteine ​​e ultracentrifugazione attraverso un cuscino di saccarosio. Poliacrilammide gel elettroforesi, colorazione Coomassie blu e occidentale recupero blotting confermato di aste prioni altamente arricchito nella frazione pellet. Aste prioni sono stati marcati con fluorocromi intraperitoneale poi iniettato in topi. Due ore dopo, cellule immunitarie dal liquido di lavaggio peritoneale, nella milza e nei linfonodi mediastinici e mesenterici sono stati analizzati per la ritenzione del prione asta e sottoinsiemi di cellule identificate mediante citometria di flusso multicolore utilizzando marcatori per i monociti, i neutrofili, cellule dendritiche, macrofagi e delle cellule B e T. Questo test permette di monitorare in tempo prima diretta di cellule immunitarie acquisizione e il traffico di prioni in vivo poche ore dopo l&#39;infezione. Questo test inoltre differenzia chiaramente infettive, aggregati da prioni PrPC normalmente espressa sulle cellule ospite, che può essere difficile e portare a problemi di interpretazione dei dati nei sistemi di dosaggio altri. Questo protocollo può essere adattato alle altre vie di inoculazione (orale, endovenosa, intranervous e sottocutaneo, ad esempio) e gli antigeni (perline coniugato, agenti patogeni batterici, virali e parassitarie e proteine ​​dell&#39;uovo) pure.

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

Qui si descrive un nuovo metodo di analisi per monitorare l&#39;assorbimento del prione e del traffico da parte delle cellule immunitarie subito dopo l&#39;inoculazione intraperitoneale purificando e fluorescente etichettatura aste prioni aggregati da materiale cerebrale infetto allora monitoraggio loro adozione, e il movimento dal sito di iniezione e caratterizzare le cellule che mediano questi eventi.

Monitoraggio Traffico di cellule immunitarie fluorescente Rods prioni ore dopo l&#39;infezione intraperitoneale

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

monitoring-immune-cells-trafficking-fluorescent-prion-rods-hours

Research

JoVE Journal

Immunology and Infection

11.4K Views.  Colorado State University. Qui si descrive un nuovo metodo di analisi per monitorare l'assorbimento del prione e del traffico da parte delle cellule immunitarie subito dopo l'inoculazione intraperitoneale purificando e fluorescente etichettatura aste prioni aggregati da materiale cerebrale infetto allora monitoraggio loro adozione, e il movimento dal sito di iniezione e caratterizzare le cellule che mediano questi eventi.

Video: Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Misurazione dell&#39;attività Calpain nelle celle fissi e Living in citometria a flusso

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Ricerca

Immunologia e infezioni

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sette passi per Stellate Cellule

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Analisi Citometria a flusso di cellule immunitarie all&#39;interno aorte Murine

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Isolamento di cellule cardiache funzionali immunitario

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

A seguito di Cell-destino<em&gt; E. coli</em&gt; Dopo l&#39;infezione da fago lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Flusso Isolamento citometrica di primari murini di tipo II cellule alveolari epiteliali per gli studi funzionali e molecolari

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Isolamento di tessuto adiposo cellule immunitarie

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

Generazione di Linfonodo grassi Pad Chimere per lo studio del linfonodo stromale origine cellulare

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Rilevamento di nanoparticelle fluorescenti Interazioni con Primaria immunitario sottopopolazioni cellulari mediante citometria di flusso

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Un approccio semi-automatico di preparare anticorpi cocktail per l&#39;analisi immunofenotipica di Human sangue periferico

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...