D.E. and M.B.D. are members of a multidisciplinary research platform (MRP) of Ghent University, and the Ghent Researchers on Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium.

In questo studio, sono stati utilizzati per adulti (6-8 settimane) femmina C57BL / 6 topi. I topi sono stati ospitati e allevati secondo le linee guida del vivaio Università di Gand. Tutte le procedure di animali sono stati approvati dalla cura degli animali e Comitato Etico Istituzionale. 1. Preparazione di cellule mononucleate (MNC) da Spleen mouse <ol><li> Sacrifica il mouse per dislocazione cervicale. </li><li> Posizionare il mouse verso il basso sul tavolo di dissezione e fissare la cerva e zampe anteriori con perni. </li><li> Sterilizzare la pelle-superficie con il 70% (v / v) di etanolo / H 2 O. </li><li> Tagliare la pelle lungo la linea mediana addominale al torace ed esporre la milza con strumenti chirurgici sterili. </li><li> Dopo il taglio dei tessuti grassi circostanti, posizionare la milza in una piastra da 24 pozzetti contenenti 1 ml di 1x tampone fosfato salino (PBS) a temperatura ambiente. </li><li> Sezionare la milza con un bisturi sterile e trasferire i pezzi in un filtro di nylon 70 micron collocato all&#39;interno di una vasca 50 mle. </li><li> Usando una 2 ml stantuffo della siringa, schiacciare delicatamente i pezzi milza attraverso il filtro delle cellule e lavare con 5 ml di PBS 1X. </li><li> Aggiungere 3 ml di Ficoll-Paque in una provetta da 15 ml e sovrapporre il mezzo gradiente di densità con la sospensione cellulare 5 ml. </li><li> Centrifugare a 400 xg per 20 min a temperatura ambiente senza freno. </li><li> Trasferire il interfase a un nuovo tubo da 15 ml, aggiungere 10 ml di freddo PBS 1X e centrifugare a 300 xg per 10 minuti a 4 ° C. </li><li> Risospendere le cellule in 2 ml di PBS 1x contenente 0,5% di albumina di siero bovino (BSA) e 1 mM di acido etilendiamminotetraacetico (EDTA) (tampone FACS). Trasferire 10 ml di sospensione cellulare in un piccolo tubo, mescolare con 10 ml di 0,4% </li></ol> 2. Arricchimento, rilevamento e purificazione delle cellule del mouse iNKT <ol><li> Arricchimento di topo milza CD5positive (CD5 +) linfociti. <ol><li> (Opzionale) Stain 10 5 cellule con FITC-coniugato anti-CD5 (4 ml di 1:30 diluizione / 10 6 celle)per 30 minuti a 4 ° C al buio, lavare con 200 ml FACS buffer e risospendere in 500 microlitri di buffer FACS. Preparare una soluzione di lavoro 20 mM di 4 &#39;, 6-diamidino-2-fenilindolo (DAPI) in PBS 1x, aggiungere 1 ml della stessa a 10 5 cellule in tampone FACS e incubare a temperatura ambiente per 5 min. Acquisire 10 4 cellule pre-arricchito sul citofluorimetro. <ol><li> Utilizzando FCS-H e FCS-W, cancello elettronico su cellule Lo FSC W di escludere doppietti cellulari e aggregati (Figura 1A). Allora cancello elettronico fuori DAPI + (morti), le cellule all&#39;interno del FSC W lo popolazione (Figura 1B), e utilizzano SSC-A e FCS-A per selezionare elettronicamente la popolazione linfocitaria per dimensione (Figura 1C). </li><li> Utilizzando cellule all&#39;interno del gate linfocitario, tracciare SSC-A contro CD5 e elettronicamente cancello CD5 + linfociti come mostrato nella Figura 1D. Acquisire il resto del campione pre-arricchito sul citometro a flusso. </li&gt; </ol></li><li> Regolare conta delle cellule a 10 7 cellule per 90 microlitri di buffer di FACS e aggiungere 10 microlitri anti-CD5 microsfere. </li><li> Incubare a 4 ° C per 15 minuti, lavare una volta con 4 ml FACS tampone e risospendere in 500 microlitri di buffer FACS freddo. Arricchisci per CD5 + linfociti con separazione delle cellule magnetico (MACS) colonne secondo le raccomandazioni del fabbricante. </li><li> Opzionale) Macchia 10 5 cellule con FITC-coniugato anti-CD5 (4 l di diluizione 1:30 / 10 6 celle) e DAPI come in 2.1.1. Acquisire 10 4 linfociti arricchite sul citofluorimetro e verificare che i varchi elettronici creati al punto 2.1.1. selezionare CD5 + linfociti nel frazione cellulare arricchito. Acquisire 10 5 cellule sul citometro a flusso e analizzare la percentuale CD5 + linfociti presenti nella frazione arricchita come mostrato nella Figura 1E - H. </li></ol></li><li> Rilevamento e isolamento di topo iNKT cells di FACS. <ol><li> Risospendere CD5 + linfociti arricchiti ad una concentrazione di circa 10 6 cellule per 20 microlitri tampone FACS contenente in anti-CD16 / CD32 (4 ml di diluizione 1:50 / 10 6 cellule) e αGalCer PE-coniugato / CD1d tetramero (1 mg / 10 6 cellule). </li><li> Incubare per 30 minuti a RT al buio. </li><li> Diluire anticorpi murini (mAbs) anti-CD3ε V500 (4 ml di 1:20 diluizione / 10 6 celle), anti-CD19 E450 (4 l di diluizione 1:50 / 10 6 celle) e anti-CD8α E450 (4 ml di 1:50 / 10 6 cellule) in tampone FACS, aggiungi CD5 + linfociti e incubare per 30 min a 4 ° C al buio. </li><li> Lavare le cellule con 1 ml tampone FACS per centrifugazione a 300 xg per 10 min a 4 ° C e risospendere in 4 ml di tampone FACS ad una concentrazione finale di 10 7 cellule / ml. </li><li> Filtrare le cellule attraverso un colino cella 30 micron e posizionare le cellule in ghiaccio. </li><li> Calibrare il citofluorimetro per la cella di smistamento secondo le raccomandazioni del costruttore 14,15. Nota: Calibrazione di un citometro di flusso per l&#39;ordinamento delle cellule richiede una formazione e può essere eseguita da un operatore esperto, o un tecnico specializzato. </li><li> Aggiungere 10 ml di una soluzione di lavoro 20 pM di DAPI per 10 6 cellule e incubare a temperatura ambiente per 5 min. Acquisire circa 10 4 CD5 + linfociti arricchito sul citofluorimetro. Elettronicamente cancello cellule Lo FSC-W escludere doppietti cellulari e aggregati (Figura 2A). Allora cancello elettronico fuori DAPI + CD8 + CD19 + cellule (canale di scarico) all&#39;interno del FSC W lo popolazione (Figura 2B), e utilizzare SSC-A e FCS-A per selezionare elettronicamente linfociti (Figura 2C), come descritto nella 2.1.1.1 . <ol><li> Trama αGalCer / CD1d tetramero da CD3ε ed elettronicamente cancello αGalCer / CD1dcellule tetramero + CD3ε + iNKT come mostrato nella Figura 2D. Cellule presenti nella frazione cellulare arricchita acquistare più del 2 x 10 5 CD5 + linfociti arricchiti per determinare la percentuale αGalCer / CD1d tetramero + CD3ε + iNKT. </li></ol></li><li> Utilizzando i varchi elettronici creati in 2.2.7., Le cellule FACS sorta αGalCer / CD1d tetramero + CD3ε + iNKT sul citofluorimetro a 70 psi utilizzando un ugello di 70 micron ad un flusso di &#39;1.5&#39;. Raccogliere le cellule iNKT ordinati in una provetta contenente 1 ml FACS 100% FCS. Successivamente lavare cellule iNKT filtrate dal lato del tubo FACS utilizzando 10 ml RMPI 1640 e centrifugare le cellule a 300 xg per 10 min a 4 ° C. </li><li> Risospendere allineati cellule iNKT in 1 ml RPMI 1640 medium e acquisire 40 microlitri dello stesso su un citofluorimetro per valutare la purezza delle cellule iNKT ordinati. Selezionare FSC-W lo DAPI - linfocitis come descritto in 2.2.7. (Figura 2E - G) ed elettronicamente cancello αGalCer / CD1d cellule tetramero + CD3ε + iNKT come mostrato nella Figura 2H. </li></ol></li></ol> 3. Cultura ed espansione di cellule iNKT mouse Giorno 0 <ol><li> Cappotto una piastra da 96 pozzetti a fondo piatto con purificata anti-CD3ε (3 mg / ml) per pozzetto per 1 ora a temperatura ambiente. Lavare due volte con 200 microlitri di PBS 1x e una volta con 200 microlitri completa terreno RPMI 1640 (10% vol / vol FCS, 100 U / ml di penicillina-streptomicina, 5.5 mM β-mercaptoetanolo). </li><li> Contare il numero di cellule vitali filtrate iNKT (fase 2.2.8) utilizzando un emocitometro 0,4% come 1.11., Centrifugare a 300 xg per 10 min a 4 ° C e risospendere a 5 x 10 5 cellule / ml in RPMI completo 1640 contenente ricombinante IL-2 murino (10 ng / ml), ricombinante murino IL-12 (1 ng / ml) e solubile CD28 anti-topo (5 mg / ml). Piastreorted cellule iNKT a 10 5 cellule / pozzetto in piastre rivestite anti-CD3ε e incubare a 37 ° C in un incubatore CO 2 per 2 giorni. </li></ol> Giorno 2 <ol start="3"><li> Trasferire le cellule ad una non rivestito 96 pozzetti a fondo piatto fresco e coltura delle cellule in condizioni di riposo in completo RPMI 1640 medium a 37 ° C in un incubatore CO 2 per 2 giorni. </li></ol> Giorno 4 <ol start="4"><li> Rimuovere le cellule dalla piastra delicatamente pipettando e trasferire le cellule a una nuova provetta da 15 ml. Centrifugare a 300 xg per 10 min a 4 ° C, risospendere a 5 x 10 5 cellule / ml in RPMI completo 1640 medium contenente ricombinante IL-7 murina (5 ng / ml), e la piastra 10 5 cellule / pozzetto in una piatta fondo 96 pozzetti per 4 giorni a 37 ° C in un incubatore CO 2. </li></ol> Giorno 8 <ol start="5"><li> Raccogliere le cellule in una nuova provetta, centrifugare a 300 xg per 10 min a 4 ° C e risospendere a 5 x 10 5 cellule / ml in RPMI completo 1640 medium contenente solubile anti-CD28 (5 mg / ml). </li><li> Cappotto una piastra da 96 pozzetti a fondo piatto con purificata anti-CD3ε (3 mg / ml) per bene e lavare come in 3.1. Piatto 10 5 cellule / pozzetto in piastre anti-CD3ε rivestiti e incubare a 37 ° C in un incubatore CO 2 fino al giorno 11. </li></ol> Giorno 11-18 <ol start="7"><li> Ripetere i passaggi 3,4-3,6. </li></ol> Giorno 19-20 <ol start="8"><li> Trasferire le cellule ad un nuovo tubo 15 ml, centrifugare a 300 xg e risospendere a 5 x 10 5 cellule / ml in RPMI 1640 medium completo. </li><li> Cellule piastra a 10 5 cellule / pozzetto in una piastra da 96 pozzetti a fondo piatto e permettere alle cellule di riposare per 2 giorni a 37 ° C in un incubatore CO 2 prima dell&#39;uso in un esperimento. </li></ol> 4. citochine da parte delle cellule iNKT mouse usando una CD1d Plate-bound Assay <ol><li> Cappotto una piastra da 96 pozzetti a fondo piatto con 100 ml di murino ricombinante CD1d (10 mg / ml in PBS 1x) per pozzetto e incubare per 1 ora a 37 ° C. </li><li> Lavare quattro volte con 200 microlitri di PBS 1x, aggiungere 200 ml di 2% FCS in PBS 1x (soluzione bloccante) per pozzetto ed incubare per 2 ore a 37 ° C. </li><li> Rimuovere la soluzione di saturazione, aggiungere 200 ml di αGalCer (5 ng / mL) per ogni bene, e incubare la piastra a 37 ° C per 16 ore. [Per la preparazione della soluzione αGalCer, aggiungere 1.085 μ 1x PBS a 55 mg di αGalCer sciolto in mezzo contenente 96 mg / ml di saccarosio, 10 mg / ml di sodio desossicolato e 5 mg / ml di Tween 20]. </li><li> Dopo l&#39;incubazione, scartare la soluzione αGalCer e lavare il ben due volte con 200 microlitri PBS 1x seguito da 200 ml completo RPMI 1640 medie. </li><li> Raccogliere le cellule iNKT espanse in vitro a giorno 21, centrifugare le cellule a 300 xg per 10 min a 4 ° C e risospendere in completa RPMI 1640 ad una concentrazione di 0,5 x 10 6 cellule / ml. </li><li> Aggiungere 200 ml di risospensione delle cellule iNKT per pozzetto e incubare per 72 ore a 37 ° C in un incubatore CO 2. </li><li> Raccogliere il surnatante e misura citochine di interesse da parte di immunoassorbimento enzyme-linked assay (ELISA) secondo il protocollo del produttore. </li></ol>

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+CD1d-restricted+repertoire+is+dominated+by+a+few+autoreactive+T+cell+receptor+families.">The mouse CD1d-restricted repertoire is dominated by a few autoreactive T cell receptor families.</a> J Exp Med. 193, 893-904 (2001).</li><li>Zhou, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+glycosphingolipid+recognition+by+NKT+cells.">Lysosomal glycosphingolipid recognition by NKT cells.</a> Science. 306, 1786-1789 (2004).</li><li>Cardell, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD1-restricted+CD4++T+cells+in+major+histocompatibility+complex+class+II-deficient+mice.">CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice.</a> J Exp Med. 182, 993-1004 (1995).</li><li>Kawano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD1d-restricted+and+TCR-mediated+activation+of+valpha14+NKT+cells+by+glycosylceramides.">CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides.</a> Science. 278, 1626-1629 (1997).</li><li>Drennan, M. B., Aspeslagh, S., Elewaut, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invariant+natural+killer+T+cells+in+rheumatic+disease:+a+joint+dilemma.">Invariant natural killer T cells in rheumatic disease: a joint dilemma.</a> Nat Rev Rheumatol. 6, 90-98 (2010).</li><li>Terabe, M., Berzofsky, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKT+cells+in+immunoregulation+of+tumor+immunity:+a+new+immunoregulatory+axis.">NKT cells in immunoregulation of tumor immunity: a new immunoregulatory axis.</a> Trends Immunol. 28, 491-496 (2007).</li><li>Molling, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+sustained+expansion+of+mouse+spleen+invariant+NKT+cell+lines+with+preserved+cytokine+releasing+capacity.">Generation and sustained expansion of mouse spleen invariant NKT cell lines with preserved cytokine releasing capacity.</a> J Immunol Methods. 322, 70-81 (2007).</li><li>Chiba, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+reliable+generation+of+invariant+natural+killer+T-cell+lines+in+vitro.">Rapid and reliable generation of invariant natural killer T-cell lines in vitro.</a> Immunology. 128, 324-333 (2009).</li><li>Gumperz, J. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+TCRs+recognize+murine+CD1.">Diverse TCRs recognize murine CD1.</a> J Immunol. 162, 161-167 (1999).</li><li>Watarai, H., Nakagawa, R., Omori-Miyake, M., Dashtsoodol, N., Taniguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+detection,+isolation+and+culture+of+mouse+and+human+invariant+NKT+cells.">Methods for detection, isolation and culture of mouse and human invariant NKT cells.</a> Nature protocols. 3, 70-78 (2008).</li><li>Houtz, B., Trotter, J., Sasaki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> BD FACService TECHNOTES. 9 (4), (2004).</li><li>Osborne, G. 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The authors have no competing interests to disclose.

Passaggi critici nel protocollo corrente comprendono l&#39;isolamento e successivo arricchimento di linfociti CD5 + (Sezione 1 e 2), FACS ordinamento (sezione 3) e il fasciame iniziale di cellule iNKT (sezione 4). Dei provvedimenti nella Sezione 1, ricordarsi di strato con attenzione la sospensione cellulare milza nel medio gradiente di densità tale che un interfasica cellulare distinta viene generato dopo la centrifugazione. La successiva arricchimento per CD5 + linfociti di separazione cellulare...

Murino invarianti natural killer T (iNKT), le cellule sono una popolazione distinta di linfociti T innate selezionati nel timo by-CD1d esprimendo timociti corticali 1,2. iNKT cellule esprimono un recettore delle cellule T (TCR) costituito da una catena invariante Vα14-Jα18 TCR accoppiato con entrambi Vβ8, Vβ7 o Vβ2 TCR 3, che è in grado di riconoscere endogena così come antigeni estranei lipidi nel contesto CD1d. Per esempio, le cellule murine iNKT riconoscono e sono attivati ​​da un antigene chiamato isoglobotrihexosylceramide lipidica endogena (iGb3) 4, nonché α-galactosylceramide (αGalCer) 5,6, un glicolipide isolato da spugne marine. TCR-dipendente attivazione delle cellule iNKT promuove il priming delle risposte immunitarie adattative, e come risultato, le cellule iNKT hanno dimostrato di essere funzionalmente coinvolti nel miglioramento o lo sviluppo di una serie di patologie comprese malattia reumatica 7 e cancro &lt;sup&gt; 8. Attualmente, ligandi di cellule iNKT sintetiche costituiscono promettenti nuovi coadiuvanti che possono essere in grado di regolare una serie di condizioni immunopatologiche. È stato precedentemente dimostrato che le cellule iNKT possono essere generati in vitro dopo isolamento dal tessuto del mouse tuttavia; molti di questi studi impiegano l&#39;uso di cellule primarie presentanti l&#39;antigene (APC) e / o linee cellulari 9, Vα14 TCR transgenici (Tg) topi 10, timomi o per la generazione di cellule derivate iNKT ibridomi 11,12. Inoltre, un gran numero di topi, elevati volumi di reagenti, quali dimeri CD1d αGalCer-caricati, e tempi lunghi di cultura fanno alcuni protocolli pubblicati meno eticamente ed economicamente accattivante 9,13. In questo rapporto si descrive un metodo adatto per l&#39;isolamento e l&#39;espansione delle cellule iNKT vitro da topo milza. Più specificamente, il protocollo descrive un metodoper arricchire le cellule iNKT da milza del mouse che riduce i topi, i reagenti e il tempo richiesto per l&#39;ordinamento delle cellule FACS, e propone un approccio ottimizzato per espandere le cellule iNKT milza ordinati in vitro.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-2</td>
			<td>eBiosciences</td>
			<td>14-8021</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-12</td>
			<td>eBiosciences</td>
			<td>39-8122-65</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-7</td>
			<td>eBiosciences</td>
			<td>14-8071</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD3e (145-2C11)</td>
			<td>eBiosciences</td>
			<td>16-0032-86</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD28 (37.51)</td>
			<td>eBiosciences</td>
			<td>16-0281-85</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD19 (eBio1D3)</td>
			<td>eBiosciences</td>
			<td>48-0193-82</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD8&#945; (53-6.7)</td>
			<td>eBiosciences</td>
			<td>48-0081-82</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-mouse CD5 (53-7.3)</td>
			<td>eBiosciences</td>
			<td>11-0051-81</td>
			<td></td>
		</tr>
		<tr>
			<td>V500-conjugated anti-mouse CD3&#949; (500A2)</td>
			<td>BD biosciences</td>
			<td>560771</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD5 (Ly-1) microbeads</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-049-301</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD16/32 (2.4G2)</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4% (wt/vol)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>12633-020</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010-056</td>
			<td>[Ca2+/Mg2+ - free]</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>EDS-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque plus</td>
			<td>GE Healthcare</td>
			<td>71-7167-00 AF</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate F-bottom</td>
			<td>Greiner-bio one</td>
			<td>657-160</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Greiner-bio one</td>
			<td>665-180</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Greiner-bio one</td>
			<td>655-180</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>188-271</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>227-261</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m filter</td>
			<td>Greiner-bio one</td>
			<td>542-070</td>
			<td></td>
		</tr>
		<tr>
			<td>30 &#181;m filter</td>
			<td>Millipore</td>
			<td>SVGP01050</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-jacketed CO2 incubator</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Kit</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized method for isolating and expanding invariant natural killer t cells from mouse spleen

La capacità di secernere rapidamente citochine dopo stimolazione è una caratteristica funzionale del invariante natural killer T (iNKT) linea cellulare. iNKT cellule sono quindi caratterizzati da una popolazione di cellule T innata capace di attivare e sterzo adattivo risposte immunitarie. Lo sviluppo di tecniche migliorate per la coltura e l&#39;espansione di cellule murine iNKT facilita lo studio della biologia cellulare iNKT in vitro ed in sistemi modello in vivo. Qui si descrive un procedimento ottimizzato per l&#39;isolamento e l&#39;espansione di cellule spleniche iNKT murine. Milza di topi C57BL / 6 vengono rimossi, sezionati e tese e la sospensione cellulare risultante è stratificato su supporti gradiente di densità. Dopo la centrifugazione, le cellule mononucleate spleniche (MNC) sono raccolti e CD5 positivi (CD5 +) linfociti sono arricchiti per l&#39;utilizzo di sfere magnetiche. cellule iNKT all&#39;interno della frazione CD5 + sono successivamente colorate con ^5; GalCer-caricato CD1d tetramero e purificata mediante la fluorescenza delle cellule attivate (FACS). FACS ordinati cellule iNKT sono quindi inizialmente coltivate in vitro utilizzando una combinazione di citochine ricombinanti murine e recettore delle cellule T piastriforme bound (TCR) stimoli prima di essere espanso in presenza di IL-7 murina ricombinante. Utilizzando questa tecnica, di circa 10 8 cellule iNKT possono essere generati entro 18-20 giorni di coltura, dopo di che possono essere utilizzate per saggi funzionali in vitro o in vivo per esperimenti di trasferimento nei topi.

L&#39;isolamento delle cellule mononucleari spleniche mediante gradiente di densità richiede circa 1 ora ed elimina l&#39;uso di reagenti necessari per la lisi dei globuli rossi (RBC). Un alto rendimento di cellule vitali è ottenuto utilizzando questo metodo e detriti generati durante sforzare dell&#39;organo viene rimosso. Tipicamente, la frequenza di cellule iNKT nel pool di linfociti splenici compresa tra 1 e 5% del totale di linfociti T tuttavia, questo può variare a seconda dello stato età, il sesso e la salute...

Watch this Scientific Journal Video about An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen at JoVE.com

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Un metodo ottimizzato per l&#39;isolamento ed espansione invariante Natural Killer cellule T da Spleen mouse

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

an-optimized-method-for-isolating-expanding-invariant-natural-killer

Research

JoVE Journal

Immunology and Infection

11.9K Views.  Ghent University Hospital. Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents. 

Video: An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Preparazione e utilizzo di HIV-1 CD4 infetti primaria + T-Cells come cellule bersaglio nei test di cellule Natural Killer citotossici

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

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Immunologia e infezioni

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Espansione linfociti T citotossici da sangue del cordone ombelicale che Target Citomegalovirus, virus di Epstein-Barr virus, e Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Antigen Presenting artificiale cellulare (AAPC) Attivazione mediata ed espansione delle cellule Natural Killer T

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Killer artificiale cellule presentanti l&#39;antigene (KaAPC) per una efficiente<em&gt; In Vitro</em&gt; L&#39;esaurimento delle Umana antigene-specifiche cellule T

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Derivazione di cellule T<em&gt; In Vitro</em&gt; Da cellule staminali embrionali di topo

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Flusso analisi di citometria di Natural Killer cellulare Lytic attività nel Sangue umano intero

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nuovo sistema privo di alimentatore per la produzione di massa di cellule di assassino naturali murini In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolando i linfociti del sistema immunitario intestinale piccolo per Mouse

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Quantificazione di basata su citometria a flusso multicolor dei mitocondri e lisosomi in cellule di T

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Analysis of Human Natural Killer Cell Metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

Analisi del metabolismo delle cellule dell'assassino naturale umano

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...