La ricerca riportata in questa pubblicazione è stato sostenuto dal National Cancer Institute del National Institutes of Health di cui al numero Award R01CA174385. Il contenuto è di esclusiva responsabilità degli autori e non necessariamente rappresentano le opinioni ufficiali del National Institutes of Health.

1. Reagenti Preparazione <ol><li> Preparare RPMI-PLGH miscelando 500 ml ml RPMI-1640 e 5 ciascuno di penicillina-streptomicina, L-glutamina, e la soluzione di HEPES. </li><li> Preparare R10 soluzione miscelando RPMI-PLGH con 10% siero fetale bovino (FBS). L&#39;FBS è inattivati ​​termicamente a 56 ° C per 30 minuti prima dell&#39;aggiunta. </li><li> Pre-riscaldare a 37 ° C 50 ml di RPMI-PLGH, 15 ml ml di PBS, e 15 di R10 in sterili provette coniche. </li><li> Fabbricazione di matrici di nanowells in PDMS: Il master di silicio è prodotto utilizzando fotolitografia essenzialmente come descritto in precedenza 10. Mescolare accuratamente il Sylgard 184 kit di base elastomero e catalizzatore nel rapporto 10:1 peso in una tazza usa e getta con un coltello di plastica. Degassare la miscela in una camera a vuoto per 1 ora. Versare il composto sulla matrice nanowell, sigillare con una lastra di vetro e lasciate riposare per 30 minuti. Trasferire l&#39;assemblaggio in un forno impostato a 80 ° C per 2 ore e lasciata raffreddare a temperatura ambiente per 1 ora. L&#39;elastomero sarà corre durante questo periodo e si legherà al vetrino. La slitta di vetro contenente la matrice PDMS nanowell è alzato attentamente fuori il padrone di silicio, ricoperto di nastro adesivo e conservati fino al momento dell&#39;uso futuro. </li></ol> 2. Obiettivo cellulare (T) Etichettatura <ol><li> Contare 5 milioni di cellule bersaglio di coltura cellulare utilizzando un emocitometro e il pellet in una provetta sterile 15 ml conica per centrifugazione a 340 xg per 5 min. La coltura cellulare è suddiviso il giorno precedente in 5 ml di volume totale (densità = 1 milione / ml) e coltivate in T-25 beuta (VWR). Protocolli per il conteggio delle cellule usando emocitometro sono stati descritti in dettaglio in precedenza 11 </li><li> Aspirare supporti in eccesso lasciando dietro di sé circa 50 microlitri di media. </li><li> Preparare una soluzione di lavoro di Cell Tracker Red (CTR) miscelando 0,5 microlitri magazzino CTR (1 mM) per 150 ml RPMI-PLGH (concentrazione finale 2,5 micron). Aggiungere 150 pl della soluzione di lavoro per le cellule bersaglio e miscelare accuratamente con una pipetta P200.Alternativamente, PKH 26 (concentrazione finale di 2 mM) può essere utilizzato cellule bersaglio etichette secondo le istruzioni del produttore. </li><li> Incubare a 37 ° C / 5% di CO 2 per 20 min. </li></ol> 3. Effector (E) Cell Etichettatura <ol><li> Contare 5 milioni di cellule effettrici di coltura cellulare utilizzando un emocitometro e il pellet in una provetta sterile 15 ml conica per centrifugazione a 340 xg per 5 min. </li><li> Aspirare supporti in eccesso </li><li> Preparare una soluzione di lavoro di Vybrant soluzione DyeCycleViolet Stain con l&#39;aggiunta di 1 ml di 5 mM magazzino Violet Vybrant a 1 ml di R10 media (concentrazione finale di 5 pm). Aggiungere la soluzione di lavoro per le cellule e risospendere usando pipetta P200. Alternativamente, PKH 67 (concentrazione finale di 2 mM) può essere utilizzato cellule bersaglio etichette secondo le istruzioni del produttore. Se PKH 67 viene utilizzata, quindi SYTOX verde non deve essere utilizzato poiché sono nello stesso canale di fluorescenza. L&#39;enumerazione degli obiettivi apoptotici si effettua esclusivamente con Acolorazione nnexinV in questo caso. Nota: Se si esegue time-lapse e non misure di endpoint, coloranti UV come Violet Vybrant dovrebbe essere evitato. </li><li> Incubare a 37 ° C / 5% di CO 2 per 20 min. </li></ol> 4. Separazione di cellule vive e morte mediante gradiente di densità <ol><li> Aggiungere 6,8 ml e 6,0 ml di RPMI-PLGH al bersaglio e cellule effettrici, rispettivamente e mescolare pipettando su e giù delicatamente. </li><li> Strato 3,5 ml di Ficoll-Paque Plus per fondo di ogni provetta conica contenente bersaglio e cellule effettrici. Strato il FICOLL lentamente per garantire una uniforme di strati tra FICOLL e media. </li><li> Centrifugare entrambi i tubi a 340 xg per 30 min. senza freno e accelerazione. </li><li> Durante la centrifugazione, trasferire 7 ml di pre-riscaldato PBS ad una 4-pozzetti. Preparare matrice nanowell (fabbricato in PDMS): pulire il fondo vetrino con etanolo e ossidare il PDMS ad alta impostazione RF utilizzando ossidante plasma per 1 min. Questo passaggio sterilize dell&#39;array nanowell mentre anche rendere idrofila la PDMS. Spostare immediatamente la matrice nanowell nella piastra contenente PBS. </li><li> Trasferire la piastra per un armadio biosicurezza e aspirare PBS in eccesso. Prova del 10 ml di agar 1% nobile con l&#39;aggiunta di polvere da 100 mg di agar a 10 ml di acqua deionizzata e poi forno a microonde che per 30-45 sec. Applicare riscaldare 1% agar nobile sul bordo superiore e inferiore del vetrino che contiene l&#39;array nanowell per immobilizzare la matrice e attendere 10 min per agar per solidificare. Aggiungere 3 ml di PBS ed aspirare agar in eccesso. </li><li> Aggiungere 3 ml di R10 sulla parte superiore della matrice nanowell e lasciare equilibrare per ~ 10 min. </li><li> A seguito di Ficoll centrifugazione, aspirare 5 Mezzi ml dallo strato superiore di ciascun tubo. Fare attenzione a non aspirare il livello contenente le cellule. </li><li> Raccogliere le cellule (strato bianco) recuperando 1-2 ml dallo strato tra FICOLL e media con P1, 000 pipetta e passare a nuove provette sterili coniche. Ripetere questa operazione per entrambi i gruppi di cellule, badando a che si massimaize le cellule di recupero. </li><li> Aggiungere 3 ml di pre-riscaldato RPMI-PLGH a ciascuna provetta, pipettando e pellet per centrifugazione a 340 xg per 5 min a piena accelerazione e freno. </li><li> Aspirare supporti in eccesso, aggiungere 5 ml di pre-riscaldato RPMI-PLGH a ciascuna provetta e ripetere la centrifugazione a 340 xg per 5 min. </li><li> Aspirare supporti in eccesso e risospendere il pellet cellulare in 500 ml di R10. </li><li> Contare le celle in diretta tramite il metodo di esclusione Trypan blu su un emocitometro. </li></ol> 5. Cella di carico su Array Nanowell <ol><li> Aspirare i supporti in eccesso dalla matrice nanowell e aggiungere 2 ml di R10 fresco attraverso la matrice. Aspirare nuovamente facendo attenzione che la parte superiore della matrice nanowell non è troppo umido / secco in quanto influirà distribuzione delle cellule. </li><li> Deposito 1 x 10 5 cellule effettrici in 200 pl di R10 sulla superficie dell&#39;array nanowell (densità = 5 x 10 5 cellule / ml). </li><li> Lasciate che le cellule accontentarsi di 5 minuti e l&#39;utilizzo di uno standard di coltura controllo dei tessuti al microscopio per enAssicurarsi che la distribuzione delle cellule è auspicabile. Aggiungi più celle, se necessario. </li><li> Deposito 1 x 10 5 cellule bersaglio in 200 microlitri R10 sulla superficie dell&#39;array nanowell (densità = 5 x 10 5 cellule / ml). </li><li> Lasciate cellule accontentarsi di 5 min e utilizzando uno standard coltura tissutale microscopio controllo per assicurare che la distribuzione delle cellule è auspicabile. Aggiungi più celle, se necessario. </li><li> Sciacquare accuratamente con matrice nanowell 2 ml R10 e aspirare i supporti in eccesso. </li><li> Preparare 3 ml di pre-riscaldato R10 in una provetta da 15 ml conica sterile. Aggiungere 3 ml di acido 0,5 mM SYTOX verde Nucleic Stain (concentrazione finale 0,5 micron) e 60 pl di annessina V-Alexa Fluor 647 a preparare la soluzione di lavoro. Aggiungere la soluzione di lavoro sul 4-pozzetti attentamente assicurandosi che le cellule non vengono spostati dai pozzetti. </li><li> Incubare a 37 ° C / 5% di CO 2 per 15 min. </li></ol> 6. Imaging 1 <ol><li> Acquisire le immagini della matrice nanowell utilizzando un micr fluorescenzaoscope: In questo esempio, usiamo un Observer ZEISS Z-1 microscopio dotato di un palco motorizzata e Lambda-DG4. </li><li> Inizializzare il software e il microscopio e controllare l&#39;intensità del segnale in luce trasmessa e gli altri canali fluorescenti. Nel complesso, vi saranno cinque canali corrispondenti a: luce trasmessa, Annessina V-647 (Cy5 filtro), CTR (DsRed filtro), SYTOX verde (FITC filtro), e Vybrant Violet (DAPI filtro). Assicurarsi che il microscopio è configurato per garantire massimo segnale al rumore, evitando la saturazione. </li><li> Impostare la posizione X e Y per la matrice nanowell. Inizia processo fissando la posizione di zero e l&#39;inizializzazione di messa a fuoco sul centro di 7 x 7 blocco di matrice nanowell in alto a sinistra del timbro matrice nanowell. Impostare le posizioni x, y in coincidenza con il centro di ogni blocco e il valore z per riflettere con precisione la messa a fuoco su ogni blocco. </li><li> Una volta che la posizione di messa a fuoco e sono tutti insieme, assicurarsi di impostare l&#39;acquisizione di immagini in modalità lineare e acquisire immagini. <sTrong&gt; Nota: Se necessario, ruotare il termostato e CO 2 sul microscopio, almeno 30 minuti prima dell&#39;esperimento. </li></ol> 7. Post-imaging Trasferire il 4-pozzetti contenente la matrice nanowell in un incubatore (37 ° C / 5% CO 2) per 6 ore. 8. Imaging 2 Acquisire le immagini al secondo time-point, come indicato al punto 6. 9. Image Analysis <ol><li> L&#39;uso di cellule all&#39;interno del nanowells viene determinato mediante l&#39;uso di opportuni programmi di segmentazione di immagini (ad es ImageJ, <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ) essenzialmente come descritto in precedenza 9,12. </li><li> Analisi della prima serie di immagini al microscopio (t = 0 hr; t 0) è usata per generare un database dei nanowells individuali contenenti esattamente una cellula bersaglio vivo (identificatoda Red CellTracker, rosso) e non le cellule morte (identificati mediante colorazione annessina V, verde) [0 T1T nanowells]. </li><li> Analisi della seconda serie di immagini al microscopio (t = 6 hr; t 6) è utilizzato per determinare indipendentemente un secondo database di nanowells contenenti 1 cella di destinazione (rosso) [T1T nanowells 6]. Un controllo di integrità (query di database) è implementato per assicurare che tutti i bersagli in scena a t 0 sono abbinati ad obiettivi a t 6 per la produzione di una serie finale di obiettivi corrispondenti (implementato come nanowells che hanno T1T 0 = T1T 6, designato come T1match) </li><li> Il numero di cellule effettrici in nanowells è determinato usando colorazione Vybrant Violet a t 0 [nanowells E1] </li><li> Una query di database viene implementato per identificare nanowells contenenti effettori singoli co-incubate con singoli target (definito come nanowells che hanno partita E1 e T1, designati come E1T1) </li><li> Effector apoptosi indotta è segnato da i<html> dentifying obiettivi che sono annessina V con negativo a t 0 e annessina V positivo t 4 (E1T1 colorazione con annessina V a destinazione t 6). </li></ol> 10. Opzionale time-lapse imaging of Killing Utilizzando Nikon BioStation IM <ol><li> Prendere una capsula di Petri con fondo coprioggetto e tagliare un piccolo pezzo del chip matrice nanowell (preparato al punto 5) che si adattano perfettamente al coprioggetto. Assicurarsi che il chip rimane bagnata durante il taglio. </li><li> Mix 1 x 10 6 obiettivi e 1 x 10 6 cellule T in 100 ul dei media, e la pipetta sul vetrino coprioggetti. </li><li> Lasciare circa 15-30 minuti per consentire alle cellule stabilirsi. </li><li> Rapidamente invertire il piccolo chip di matrice nanowell e posizionarlo sul coprioggetto. </li><li> Al fine di garantire che il chip non sarà in grado di passare nel luogo di imaging, uno o due 20mm x 20 millimetri coprioggetti sulla sommità del chip. Applicare pressione per spingere dolcemente il chip al fondo del piatto. </li></ol>tenda &quot;&gt; Nota: in basso Il chip matrice nanowell è troppo spessa per imaging ad alta risoluzione e deve quindi essere capovolto Durante questo, molte delle cellule vengono lavati Dal momento che questo non può essere facilmente controllate, il numero di cellule al punto 9.2.. e il tempo di incubazione nel passo 9,3 devono essere ottimizzati.

<ol>
	<li>Louis, C. U., 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=Antitumor+activity+and+long-term+fate+of+chimeric+antigen+receptor-positive+T+cells+in+patients+with+neuroblastoma.">Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma.</a> Blood. 118, 6050-6056 (2011).</li><li>Savoldo, B., 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=CD28+costimulation+improves+expansion+and+persistence+of+chimeric+antigen+receptor-modified+T+cells+in+lymphoma+patients.">CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients.</a> J. Clin. Invest. 121, 1822-1826 (2011).</li><li>Kalos, M., 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=T+cells+with+chimeric+antigen+receptors+have+potent+antitumor+effects+and+can+establish+memory+in+patients+with+advanced+leukemia.">T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.</a> Sci. Transl. Med. 3, 95ra73 (2011).</li><li>Porter, D. L., Levine, B. L., Kalos, M., Bagg, A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+Antigen+Receptor-Modified+T+Cells+in+Chronic+Lymphoid+Leukemia.">Chimeric Antigen Receptor-Modified T Cells in Chronic Lymphoid Leukemia.</a> N. Engl. J. Med. , (2011).</li><li>Restifo, N. P., Dudley, M. E., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+immunotherapy+for+cancer:+harnessing+the+T+cell+response.">Adoptive immunotherapy for cancer: harnessing the T cell response.</a> Nat. Rev. Immunol. 12, 269-281 (2012).</li><li>Pittet, M. J., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging.">Intravital imaging.</a> Cell. 147, 983-991 (2011).</li><li>Sumen, C., Mempel, T. R., Mazo, I. B., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy:+visualizing+immunity+in+context.">Intravital microscopy: visualizing immunity in context.</a> Immunity. 21, 315-329 (2004).</li><li>Lamoreaux, L., Roederer, M., Koup, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+cytokine+optimization+and+standard+operating+procedure.">Intracellular cytokine optimization and standard operating procedure.</a> Nat. Protoc. 1, 1507-1516 (2006).</li><li>Varadarajan, N., 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=A+high-throughput+single-cell+analysis+of+human+CD8++T+cell+functions+reveals+discordance+for+cytokine+secretion+and+cytolysis.">A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis.</a> J. Clin. Invest. 121, 4322-4331 (2011).</li><li>Ogunniyi, A. O., Story, C. M., Papa, E., Guillen, E., Love, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+individual+hybridomas+by+microengraving+to+discover+monoclonal+antibodies.">Screening individual hybridomas by microengraving to discover monoclonal antibodies.</a> Nat. Protoc. 4, 767-782 (2009).</li><li>Streeck, H., Frahm, N., Walker, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+IFN-gamma+Elispot+assay+in+HIV+vaccine+research.">The role of IFN-gamma Elispot assay in HIV vaccine research.</a> Nat. Protoc. 4, 461-469 (2009).</li><li>Varadarajan, N., 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,+efficient+functional+characterization+and+recovery+of+HIV-specific+human+CD8++T+cells+using+microengraving.">Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 3885-3890 (2012).</li><li>Makedonas, G., Betts, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+in+a+house+of+cards:+re-evaluating+CD8++T-cell+immune+correlates+against+HIV.">Living in a house of cards: re-evaluating CD8+ T-cell immune correlates against HIV.</a> Immunol. Rev. 239, 109-124 (2011).</li><li>Telford, W. G., Komoriya, A., Packard, B. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+analysis+of+apoptosis+by+flow+and+image+cytometry.">Multiparametric analysis of apoptosis by flow and image cytometry.</a> Methods Mol. Biol. 263, 141-160 (2004).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo delineato il protocollo per un high-throughput cella singola saggio citolitica abilitato tramite co-incubazione di effettori e degli obiettivi in matrici di nanowells (Figura 1). Oltre a un throughput un grande vantaggio di questa tecnica è la capacità di monitorare effettore la citotossicità contro cellule bersaglio desiderate senza necessità di engineering cellule bersaglio che a sua volta consente l&#39;uso di cellule tumorali autologhe o matched / primaria come cellule bersaglio. Il conf...

<table border="1"><tbody><tr><td> Nome del reagente / materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> RPMI-1640 w / o L-glutammina </td><td> Cellgro </td><td> 15-040-CV </td><td></td></tr><tr><td> Penicillina-streptomicina </td><td> Cellgro </td><td> 30-002-CI </td><td> 10.000 UI penicillina 10.000 mg / ml streptomicina </td></tr><tr><td> L-glutammina </td><td> Cellgro </td><td> 25-005-CI </td><td> 200 mM soluzione </td></tr><tr><td> HEPES </td><td> Sigma Aldrich </td><td> H3537 </td><td> 1M </td></tr><tr><td> Siero bovino fetale (FBS) </td><td> Atlanta Biologicals </td><td> S11150 </td><td> Lotto testato </td></tr><tr><td> Cella Tracker Red Stain </td><td> Invitrogen </td><td> C34552 </td><td> 50 ug </td></tr><tr><td> Vybrant DyeCycle Violet Stain </td><td> Invitrogen </td><td> V35003 </td><td> 5 mM </td></tr><tr><td> SYTOX acidi nucleici verde Stain </td><td> Invitrogen </td><td> S7020 </td><td> 5 mM </td></tr><tr><td> Annessina V-Alexa Fluor 647 </td><td> Invitrogen </td><td> A23204 </td><td> 500 pl </td></tr><tr><td> Dulbecco PBS </td><td> Cellgro </td><td> 21-031-CV </td><td> 500 ml </td></tr><tr><td> Agar Noble </td><td> DIFCO </td><td> 2M220 </td><td> 100 g </td></tr><tr><td> Trypan Blu </td><td> Sigma Aldrich </td><td> T8154 </td><td> 0,4% di liquido, filtrato sterile </td></tr><tr><td> Emocitometro </td><td> Hausser Scientifics </td><td> 1492 </td><td> Linea luminosa </td></tr><tr><td> 4 pozzetti </td><td> Thermo Fisher </td><td> 167603 </td><td></td></tr><tr><td> Harrick Plasma Cleaner </td><td> Harrick Plasma </td><td> PDC-32G </td><td> Di base Plasma cleaner </td></tr><tr><td> Observer.Z1 </td><td> ZEISS </td><td&gt; </td><td> Microscopio a fluorescenza (funziona con le tre parti di seguito) </td></tr><tr><td> Lambda 10-3 </td><td> Sutter Instrument </td><td></td><td> Filtro regolatore </td></tr><tr><td> Lambda DG-4 </td><td> Sutter Instrument </td><td></td><td> Ultra-High-Speed ​​Lunghezza d&#39;onda switcher </td></tr><tr><td> Hamamatsu EM-CCD </td><td> Hamamatsu </td><td> C9100-13 </td><td> Microscopio fotocamera CCD- </td></tr><tr><td> 15 ml tubo conico </td><td> BD Falcon </td><td> 352097 </td><td></td></tr><tr><td> Provetta da 50 ml </td><td> VWR </td><td> 3282-345-300 </td><td></td></tr><tr><td> Nikon Biostation </td><td> Nikon Instruments Europe BV </td><td> Biostation IM </td><td></td></tr><tr><td> Con fondo di vetro piatto di coltura </td><td> MatTek Corporation </td><td> P35G-0 </td><td> 35 millimetri Petri, 10 mm micropozzetti </td></tr></tbody></table>

quantitative high-throughput single-cell cytotoxicity assay for t cells

Immunoterapia cancro può sfruttare la specificità della risposta immunitaria per individuare e eliminare i tumori. Terapia cellulare adottiva (ACT) basato sul trasferimento adottivo di cellule T geneticamente modificate per esprimere un recettore chimerico antigene (CAR) ha mostrato promettente in studi clinici 1-4. Ci sono molti vantaggi di utilizzare CAR + cellule T per il trattamento di tumori tra cui la possibilità di individuare antigeni ristrette non MHC e di funzionalizzare le cellule T per la sopravvivenza ottimale, homing e la persistenza all&#39;interno dell&#39;ospite e, infine, per indurre l&#39;apoptosi delle CAR + cellule T in caso di tossicità ospitante 5. Delineare le funzioni ottimali di CAR + cellule T associate a beneficio clinico è essenziale per la progettazione della prossima generazione di studi clinici. I recenti progressi nella diagnostica per immagini di animali vivi, come la microscopia multifotone hanno rivoluzionato lo studio della funzione delle cellule immunitarie in vivo 6,7. Mentre questi studi hanno avanzato la nostra comprensione di funzioni delle cellule T in vivo, cellule T ACT basata in studi clinici richiede la necessità di collegare le caratteristiche molecolari e funzionali di cellule T preparazioni pre-infusione con efficacia clinica post-infusione, utilizzando in vitro monitoraggio funzioni delle cellule T come, citotossicità e la secrezione di citochine. Standard di flusso-citometria saggi basati sono stati sviluppati che determinano il funzionamento complessivo di popolazioni di cellule T a livello di singola cellula, ma questi non sono adatti per il monitoraggio e la formazione coniugato vite o la capacità della cellula stessa per uccidere più bersagli 8. Array microfabbricati progettati in polimeri biocompatibili come polidimetilsilossano (PDMS) è un metodo particolarmente interessante per limitare spazialmente effettori e gli obiettivi in piccoli volumi 9. In combinazione con automatico time-lapse microscopia a fluorescenza, thousands di effettore bersaglio interazioni può essere monitorato contemporaneamente da singoli pozzetti di imaging di una matrice di nanowell. Vi presentiamo qui un high-throughput metodologia per il monitoraggio delle cellule T citotossicità mediata a livello di singola cellula che può essere ampiamente applicata allo studio della funzionalità delle cellule T citolitica.

Un esempio di applicazione del saggio ad alta prestazione citolitica è mostrato nella Figura 2. In breve, l&#39;etichetta-CD19 specifici CAR + cellule T sono state co-incubate con marcate topo EL4 cellule bersaglio nei singoli pozzetti di una matrice nanowell (sezioni 1-5). L&#39;immagine iniziale è stata registrata sul microscopio a fluorescenza automatizzata per identificare l&#39;occupazione (effettori e / o gli obiettivi) di ogni singolo nanowell sull&#39;array (sezione 6). L&#39;elabor...

Watch this Scientific Journal Video about Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells at JoVE.com

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Descriviamo una singola cellula saggio ad alta prestazione per misurare la citotossicità delle cellule T quando incubati con cellule tumorali bersaglio. Questo metodo impiega una densa matrice elastomerica di sub-nanolitri pozzetti (~ 100.000 pozzetti / matrice) per confinare spazialmente le cellule T e le cellule bersaglio con rapporti definiti ed è accoppiato al microscopio a fluorescenza per monitorare effettore-bersaglio coniugazione e apoptosi successiva.

Quantitativa High-throughput cella singola test di citotossicità per le cellule T

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

quantitative-high-throughput-single-cell-cytotoxicity-assay-for-t

Research

JoVE Journal

Immunology and Infection

14.9K Views.  University of Houston. Descriviamo una singola cellula saggio ad alta prestazione per misurare la citotossicità delle cellule T quando incubati con cellule tumorali bersaglio. Questo metodo impiega una densa matrice elastomerica di sub-nanolitri pozzetti (~ 100.000 pozzetti / matrice) per confinare spazialmente le cellule T e le cellule bersaglio con rapporti definiti ed è accoppiato al microscopio a fluorescenza per monitorare effettore-bersaglio coniugazione e apoptosi successiva.

Video: Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

High-throughput Metodo di rilevazione per il virus dell&#39;influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Ricerca

Immunologia e infezioni

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Una valutazione quantitativa della migrazione cellulare con il Test Phagokinetic motilità Traccia

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Un test fenotipico microscopico per la quantificazione dei micobatteri intracellulari adattati per lo screening ad alta produttività / alto contenuto

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

High-throughput Quantitative Real-time RT-PCR test per la determinazione di espressione Profili di tipo I e III interferone sottotipi

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Misura High Throughput di extracellulare DNA di uscita e quantitativa NET Formazione in neutrofili umani<em&gt; In Vitro</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

High Throughput, in tempo reale, Dual-lettura Prove di intracellulare antimicrobica attività e eucariotica citotossicità cellulare

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

Una fluorescenza a base di linfociti Assay Adatto per high-throughput screening di piccole molecole

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

Un'analisi di High-throughput per valutare e quantificare la trappola extracellulari del neutrofilo formazione

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Un High-throughput Shigella-specifica analisi battericida

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Funzionalizzazione dei cantilever a microscopio a forza atomica con cellule A T singole o a particella singola per la spettroscopia immunologica a forza a cella singola

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...