This work was supported by the Swedish Cancer Society (11 0253, 13 0469), the Swedish Research Council (K2012-99X-21959-01-3), Marcus Borgstr&#246;ms Foundation and the Swedish Research Council through a grant to the Uppsala RNA Research Centre (2006-5038-36531-16). We are indebted to the BioVis core facility at Uppsala University for much help with the FACS analysis.

Il protocollo descrive l&#39;isolamento di cellule linfoidi dal materiale umano paziente e richiede quindi l&#39;approvazione etica. Il lavoro svolto in questo studio è stata concessa dalla Uppsala Ethical Review Board (DNR. 2013/387). 1. Isolamento di cellule mononucleate (MNC) da umani Palatine Tonsille ATTENZIONE: Tutto il materiale non schermati di origine umana come sangue, tessuti o fluidi corporei dovrebbe essere considerato come materiale potenzialmente infetto. Pertanto, pratiche di biosicurezza consigliate per la manipolazione dei tessuti umani devono essere seguite. Nota: Per evitare contaminazioni, tutte le soluzioni e le attrezzature colture cellulari devono essere sterili. Tutti i tamponi e le soluzioni devono essere pre-raffreddati e tenuti in ghiaccio. Tessuti di tonsille e cellule isolate dovrebbero essere tenuti e gestiti su ghiaccio. <ol><li> Ottenere tonsille fresche da pazienti sottoposti a tonsillectomia o tonsillotomia (Figura 2). Tuffatevi la Clum tessutips in un tubo da centrifuga da 50 ml contenente una soluzione sterile Hanks equilibrata sale ghiacciata (HBSS) supplementato con siero fetale bovino al 5% (FBS), glutammina 10 mM, 0,05 mg / ml di gentamicina e 1% mix antibiotici contro funghi (Penicillina, streptomicina e amfotericina B). </li><li> Tenere le provette con i campioni di tessuto tonsille sommersi in ogni momento sul ghiaccio. Provare per elaborare le tonsille nel più breve tempo possibile, e comunque entro 3 ore dopo la rimozione chirurgica. </li><li> Posizionare la tonsilla su una cella di plastica piastra di coltura 60 millimetri su ghiaccio e mantenere il tessuto inumidito con HBSS. Rimuovere coaguli di sangue visibili, adiposo e tessuto connettivo con una pinza pulita dalla superficie tonsille. </li><li> Utilizzare una nuova piastra di coltura cellulare 60 millimetri contenente 5 ml di HBSS. Tagliare il ciuffo tessuto tonsillare in 3-10 frammenti mm utilizzando un paio di forbici sterili e / o un bisturi. </li><li> Preparare una nuova piastra di coltura cellulare 60 mm contenente 10 ml di HBSS e posizionare un colino plastica cella 100 micron nella soluzione HBSS. </li><li> Trasferire i dissframmenti tonsillari rifletteva nel colino cella con una pinza sterile. Usando l&#39;estremità stantuffo di una siringa di plastica, uniformemente spremere i frammenti di tessuto attraverso il filtro cella. Assicurarsi che i frammenti tonsillari sono totalmente immersi nel HBSS. </li><li> Eliminare il filtro cella con i resti di tessuto e trasferire la sospensione cellulare risultante dalla piastra di coltura cellulare 60 mm in un tubo di plastica da 50 ml centrifuga. Lasciare la provetta in ghiaccio mentre si procede con passo 1.8 e 1.9. </li><li> Nel caso di grandi tonsille, più grande di 2 cm (Figura 2), spremere i frammenti tonsille in due filtri cellulari per evitare l&#39;intasamento della rete nel filtro. </li><li> Ottenere il volume finale di 35 ml di sospensione cellulare. Nota: A questo punto è possibile preparare la sospensione di cellule per la crioconservazione (vedere la sezione 2). </li><li> Aggiungere 10 ml di soluzione di gradiente di densità Ficoll come in una nuova provetta da centrifuga da 50 ml. Sovrapporre delicatamente i suspens cellulariion (passo 1.9) sulla parte superiore della soluzione gradiente di densità. Evitare la miscelazione della sospensione cellulare e la soluzione gradiente di densità. Nota: La soluzione di gradiente di densità deve essere riscaldato a temperatura ambiente. </li><li> Centrifugare la sospensione cellulare in un rotore incernierato a 700 xg per 20 minuti a RT. Non attivare la funzione di freno alla centrifuga come frenata veloce può interrompere il gradiente. Inoltre, utilizzare la funzione di accelerazione più basso sul centrifuga. Nota: Dopo questo passo centrifugazione, osservare multinazionali come uno strato bianco soffice all&#39;interfaccia, e globuli rossi (RBC), fibroblasti e detriti cellulari come il sedimento sul fondo della provetta. </li><li> Raccogliere accuratamente lo strato multinazionali utilizzando una pipetta da 10 ml. Posizionare la sospensione cellulare in una nuova provetta da centrifuga da 50 ml. </li><li> Aggiungere 25 ml di PBS ghiacciato alla sospensione cellulare e girare il tubo a 300 xg per 5 minuti in un rotore incernierato a 4 ° C. </li><li> Rimuovere il surnatante con una pipetta 25 mle ripetere pellet cellulare lavaggio passo altre due volte. Infine risospendere il pellet cellulare in 15 ml di PBS. Nota: Dopo la fase di lavaggio finale, è possibile crioconservare la sospensione cellulare (vedi capitolo 2). </li><li> Applicare 10 ml di sospensione cellulare in camera di conteggio delle cellule emocitometro e contare il numero di cellule al microscopio. Distribuire la quantità appropriata di cellule (ad esempio, 2-3 x 10 7) in una provetta da 15 ml per centrifuga successiva separazione magnetica tallone. Tenere questo aliquota in ghiaccio fino al punto 3.2. </li></ol> 2. La crioconservazione delle cellule tonsillare <ol><li> Preparare medio di congelamento (90% FBS e il 10% DMSO) e tenerlo in ghiaccio. Per la conservazione a lungo termine mantenere i mezzi di comunicazione di congelamento a -20 ° C. </li><li> Determinare il numero totale di cellule utilizzando una camera di conteggio cellulare emocitometro (passo 1.15). Calcolare la quantità di terreno necessaria congelamento secondo la densità cellulare congelato desiderata (ad esempio, 10 7 cellules / ml di media di congelamento). </li><li> Centrifugare la sospensione cellulare a 300 xg per 5 min. Decantare il surnatante senza disturbare il pellet e risospendere il pellet cellulare in mezzo congelamento ghiacciato (dal punto 2.1). </li><li> Dispensare 1 ml aliquote della sospensione cellulare in fiale sterili progettati per lo stoccaggio a lungo termine in azoto liquido. Congelare le fiale in una camera isopropanolo e conservarli a -80 ° CO / N. Per la conservazione a lungo termine trasferire le fiale in azoto liquido contenente serbatoio o di una cella freezer -140 ° C. </li><li> Per scongelare fiale con cellule congelate, li riscaldare rapidamente in un bagno d&#39;acqua a 37 ° C. Immediatamente quando scongelato, disperdere la sospensione cellulare in 10 ml di pre-riscaldato HBSS (con supplementi, punto 1.1) in una provetta da centrifuga da 15 ml. Spin il tubo a 300 xg per 5 min, rimuovere HBSS e risospendere il pellet cellulare alla densità cellulare desiderato in HBSS (con supplementi, punto 1.1). Nota: La vitalità della multinazionali after scongelamento è di importanza, in quanto la presenza di cellule morte diminuisce la resa finale purificata B e T linfociti. Nota: Secondo la nostra esperienza la vitalità cellulare meno 80% ridurrà l&#39;efficienza isolamento delle cellule. </li></ol> 3. selezione positiva di T linfociti Popolazione da tonsillare multinazionali Nota 1: Questo protocollo si basa sulla selezione positiva di umani linfociti T CD3 + da multinazionali tonsillari utilizzando sfere magnetiche accoppiate per l&#39;anticorpo CD3. E &#39;possibile iniziare questa sezione dal fresco (sezione 1) o congelati (sezione 2) multinazionali. Nota 2: Iniziare con 3 x 10 7 multinazionali. Non superare questo numero di cellulare, dal momento che le colonne di separazione possono intasare e questo ridurrà l&#39;efficienza di isolamento. Utilizzare le colonne più grandi se verranno trattati più celle. I volumi utilizzati in questo protocollo sono state sperimentalmente ottimizzati per il numero di cellule uscato nel nostro setup sperimentale. <ol><li> Preparare il tampone di separazione [PBS (pH 7,2), lo 0,5% di albumina sierica bovina (BSA) e 2 mM EDTA]. Filtrare il tampone di separazione attraverso un filtro da 0,45 micron e conservare a 4 ° C. </li><li> Spin sospensione PTM (passo 1.14) a 300 xg per 5 minuti a 4 ° C. Risospendere il pellet risultante cellule in 240 microlitri (80 microlitri per 10 7 cellule) di tampone di separazione ghiacciato. Trasferire la sospensione cellulare in una provetta sterile da 2 ml. </li><li> Aggiungere 20 ml di anticorpo CD3 magnetica alla soluzione cellulare. Incubare le provette per 1 ora a 4 ° C con continua miscelazione delicata per mantenere le cellule in sospensione. </li><li> Trasferire tutto di sospensione cellulare in una provetta da 15 ml contenente 5 ml di tampone di separazione ghiacciata per lavare le cellule. </li><li> Centrifugare la provetta a 300 xg per 10 min a 4 ° C in un rotore incernierato. </li><li> Nel frattempo, impostare il separatore magnetico e di colonna. Fissare il separatore magnetico per il supporto e posizionare la colonna nel SEPARator. Lavare la colonna applicando 500 ml di buffer di separazione ghiacciato. Gettare il flusso attraverso. </li><li> Inserire un nuovo tubo di raccolta 15 ml sotto la colonna. Tenere il tubo di raccolta sul ghiaccio. </li><li> Eliminare il surnatante (passo 3,5) di pipetta e risospendere delicatamente il pellet cellulare in 500 microlitri tampone di separazione. Applicare la sospensione cellulare sulla sommità della colonna pre-lavata e farlo funzionare attraverso. Nota: grumi di cellule possono ostruire la colonna e ridurre la portata così. Per evitare questo problema, inserire un filtro cella 40 micron plastica sulla parte superiore della colonna. Passando le cellule attraverso la maglia sarà altamente migliorare l&#39;efficienza di questo metodo. </li><li> Lavare la colonna 4 volte con 1,5 ml di tampone di separazione (500 microlitri per 10 7 cellule). Attendere il serbatoio colonna vuota (nessun liquido dovrebbe essere osservato nella colonna) prima di applicare il lavaggio successivo passo. Nota: Ottenere le cellule CD3 non marcati, considerati come frazione di linfociti B, in quanto sonoeluito nel tubo di raccolta. </li><li> Rimuovere la colonna dal separatore e posto in un nuovo tubo di raccolta da 15 ml. Pipettare 2 ml di tampone di separazione nella colonna. Utilizzando il pistone in dotazione con la colonna eluire i linfociti T selezionati positivamente, che sono considerati come la frazione di linfociti T. </li><li> Determinare il numero di cellule purificate (passo 1.15) se necessario. Sospensioni cellulari sono ora pronti per esperimenti a valle. </li></ol> 4. Citometria a flusso Analisi dei isolata tonsillare linfociti B e T ATTENZIONE: soluzione Paraformaldeide è irritante e sospetto cancerogeno. Indossare indumenti protettivi, guanti e proteggersi gli occhi / la faccia. Nota 1: Questo protocollo descrive il metodo per la colorazione diretta del B isolata e le cellule T per fluorescenza cellulare Attivato Ordinamento (FACS) analisi. Lo scopo principale di questa fase è quello di valutare la purezza della cella isolata popolazioni after CD3 anticorpo separazione magnetica. A tal fine stabilito B e marcatori di cellule T, si utilizzano anticorpi monoclonali fluoroforo coniugato CD20-FITC e CD2-APC. Anche l&#39;inserimento di anticorpi di controllo isotipo è altamente raccomandato per distinguere la non specifico &quot;sfondo&quot; legame di anticorpi CD2 e CD20 (vedi punto 4.8). Nota 2: E &#39;possibile mantenere le frazioni cellulari purificate in paraformaldeide 1% di soluzione (PFA) al buio a 4 ° C fino al momento della colorazione (ad esempio, il giorno dopo.). Anche la stessa procedura è applicabile se esiste un intervallo di tempo tra la colorazione e l&#39;analisi FACS. Ricorda che in entrambi i casi le cellule devono essere lavati correttamente con PBSA (PBS contenente 0,2% di BSA) tampone, prima della colorazione o l&#39;analisi FACS. <ol><li> Estrarre 6 x 10 6 cellule delle multinazionali dal punto 1.15 (prima di applicare alla colonna), frazione di linfociti B dal punto 3.9 e la frazione di linfociti T a partire dal punto 3.10. </li><li> Spin la cellasospensioni a 300 xg per 5 minuti a 4 ° C con un rotore swing-out. </li><li> Rimuovere il surnatante con pipetta e lavare il pellet cellulare una volta con 1 ml di ghiacciata PBSA. Spin le sospensioni cellulari a 300 xg per 5 minuti a 4 ° C. Rimuovere il surnatante con pipetta e risospendere il cellule in 600 ml di tampone PBSA ghiacciato. </li><li> Aggiungere 100 ml di sospensione cellulare al fondo di un tubo FACS. </li><li> Aggiungere 100 ml di PBS contenente il 10% di calore siero umano inattivato a ciascuna provetta, mescolare bene e incubare per ~ 1 minuti a temperatura ambiente oppure 20 minuti in ghiaccio. Nota: i linfociti B trasportano recettori Fc. Per bloccare il recettori Fc frazioni cellulari purificate sono incubati con 10% di siero umano inattivato al calore. Il siero umano inattivato al calore è da incubazione a 56 ° C per 1 ora. Dividete il calore siero inattivato in piccole aliquote e conservare congelati a -20 ° C. </li><li> Centrifugare la sospensione cellulare a 300 xg per 5 minuti a 4 ° C in un swing-out rotore. </li><li> Rimuovere il surnatante con pipetta e lavare il pellet cellulare con 1 ml PBSA. Ripetere la centrifugazione (300 xg per 5 minuti a 4 ° C). </li><li> Aggiungere 100 microlitri PBSA in ciascun tubo in ghiaccio. Aggiungere la quantità appropriata di anticorpo monoclonale coniugato fluoroforo, secondo la raccomandazione del costruttore (ad esempio, 20 ml di anti-CD20 e / o 5 ml di anticorpi anti-CD2 in 100 ml di PBSA). Nota: Non dimenticare di assegnare provette di controllo sufficienti per l&#39;analisi FACS. In questo protocollo dovrebbero essere inclusi i seguenti controlli: 1) cellule colorate con gli anticorpi anti-CD2 e anti-CD20 individualmente, 2) cellule colorate con gli anticorpi anti-CD2 e anti-CD20 controllo isotipico singolarmente e, 3) celle senza aggiunta di anticorpi. </li><li> Brevemente vortice la provetta e incubare per 30 min a 4 ° C al buio. </li><li> Lavare 2 volte con PBSA. Aggiungere 2 ml di soluzione all&#39;1% PFA ai campioni. Lasciare le cellule rimangono nella soluzione PFA al 476; C al buio fino al momento dell&#39;analisi FACS. </li><li> Immediatamente prima di eseguire i campioni nella macchina FACS, lavare le cellule 2 volte con PBSA (300 xg per 5 minuti a 4 ° C). Campioni risospendere in 1 ml di PBS e mantenere a 4 ° C (o su ghiaccio), protetto dalla luce, prima della separazione sul citometro a flusso. </li><li> Fare l&#39;ordinamento FACS secondo il protocollo del citofluorimetro produttore. </li></ol> 5. PCR individuazione del DNA Adenovirus in isolati tonsillare linfociti B e T Nota 1: Le adenovirus hexon primer gene sono AdRJC1 (5&#39;-GACATGACTTTCGAGGTCGATCCCATGGA-3 &#39;) e AdRJC2 (5&#39;-CCGGCTGAGAAGGGTGTGCGCAGGTA-3&#39;) 11. Questi primer generano un amplicone di 139 bp. Il padrone di casa gene 18S rRNA può essere rilevato con primer tp206 (5&#39;-CCCCTCGATGCTCTTAGCTG-3 &#39;) e TP207 (5&#39;-TCGTCTTCGAACCTCCGACT-3&#39;). La dimensione prevista amplicone è di 300 bp. Nota 2: Ogni PCRcorsa comprende un controllo negativo (distillata H 2 O) e un controllo DNA positivo ottenuto da linea di cellule B umane (BJAB) infettate con il tipo di adenovirus umano 5 12. <ol><li> Estrarre il DNA (da almeno 1 x 10 6 cellule, passo 3.11) con membrana silicea metodo di purificazione basato su colonne. Estrarre RNA utilizzando fenolo e guanidina isotiocianato soluzione 13. </li><li> Aggiungere 100 ng di DNA da linfociti B e T di una miscela di reazione contenente tampone 1x HF, 0,2 mM di mix trifosfato di deossinucleotide (dNTP), 0,25 mM di ciascun primer e 1 U di alta fedeltà polimerasi DNA in un volume totale di 20 microlitri. </li><li> Eseguire amplificazione PCR con le seguenti condizioni di ciclo: 30 sec di denaturazione a 98 ° C, seguita da 30 cicli di 98 ° C per 10 sec, 67 ° C (hexon) o 58 ° C (18S rRNA) per 30 sec e 72 ° C per 30 sec. La reazione di PCR è stata conclusa con un passo estensione finale di 7 min a 72 ° C. </li><li> Separare il conseguente amplif PCRprodotti icazione su un gel di agarosio 1,5% in TBE 1x tampone e visualizzare le bande previsti dalla colorazione con colorante acido nucleico. </li></ol>

<ol>
	<li>Nave, H., Gebert, A., Pabst, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+immunology+of+the+human+palatine+tonsil.">Morphology and immunology of the human palatine tonsil.</a> Anat Embryol (Berl). 204, 367-373 (2001).</li><li>Perry, M., Whyte, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+the+tonsils.">Immunology of the tonsils.</a> Immunology today. 19, 414-421 (1998).</li><li>Alkhalaf, M. A., Guiver, M., Cooper, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+quantitation+of+adenovirus+DNA+from+human+tonsil+and+adenoid+tissues.">Prevalence and quantitation of adenovirus DNA from human tonsil and adenoid tissues.</a> J Med Virol. 85, 1947-1954 (2013).</li><li>Brandtzaeg, P., Halstensen, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+and+immunopathology+of+tonsils.">Immunology and immunopathology of tonsils.</a> Adv Otorhinolaryngol. 47, 64-75 (1992).</li><li>Imai, 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=Epstein-Barr+virus+(EBV)-carrying+and+-expressing+T-cell+lines+established+from+severe+chronic+active+EBV+infection.">Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection.</a> Blood. 87, 1446-1457 (1996).</li><li>Veen, J., Lambriex, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+adenovirus+to+lymphocytes+in+naturally+infected+human+tonsils+and+adenoids.">Relationship of adenovirus to lymphocytes in naturally infected human tonsils and adenoids.</a> Infect Immun. 7, 604-609 (1973).</li><li>Friedman, M., Wilson, M., Lin, H. C., Chang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Updated+systematic+review+of+tonsillectomy+and+adenoidectomy+for+treatment+of+pediatric+obstructive+sleep+apnea/hypopnea+syndrome.">Updated systematic review of tonsillectomy and adenoidectomy for treatment of pediatric obstructive sleep apnea/hypopnea syndrome.</a> Otolaryngol Head Neck Surg. 140, 800-808 (2009).</li><li>Garnett, C. 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=Latent+species+C+adenoviruses+in+human+tonsil+tissues.">Latent species C adenoviruses in human tonsil tissues.</a> J Virol. 83, 2417-2428 (2009).</li><li>Garnett, C. T., Erdman, D., Xu, W., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+quantitation+of+species+C+adenovirus+DNA+in+human+mucosal+lymphocytes.">Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes.</a> J Virol. 76, 10608-10616 (2002).</li><li>Perez, M. E., Billordo, L. A., Baz, P., Fainboim, L., Arana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+memory+B+cells+isolated+from+blood+and+tonsils+are+functionally+distinctive.">Human memory B cells isolated from blood and tonsils are functionally distinctive.</a> Immunol Cell Biol. 92, 882-887 (2014).</li><li>Cooper, R. J., Yeo, A. C., Bailey, A. S., Tullo, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+polymerase+chain+reaction+assay+for+rapid+diagnosis+of+conjunctivitis.">Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis.</a> Invest Ophthalmol Vis Sci. 40, 90-95 (1999).</li><li>Zhang, Y., Huang, W., Ornelles, D. A., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+adenovirus+latency+in+human+lymphocyte+cell+lines.">Modeling adenovirus latency in human lymphocyte cell lines.</a> J Virol. 84, 8799-8810 (2010).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal. biochem. 162, 156-159 (1987).</li><li>Watanabe, T., Yoshizaki, K., Yagura, T., Yamamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+antibody+formation+by+human+tonsil+lymphocytes.">In vitro antibody formation by human tonsil lymphocytes.</a> J Immunol. 113, 608-616 (1974).</li></ol>

The authors have nothing to disclose.

Uno dei fattori più importanti che influenzano il risultato di questo protocollo è l&#39;uso di materiale fresco tonsillare come materiale di partenza. Pertanto, i campioni di tonsille devono essere trattati entro 3 ore dopo l&#39;intervento chirurgico. Tonsille possono essere ottenuti da adulti e bambini. Il materiale tonsillare dai bambini è di solito più piccoli a causa della rimozione chirurgica parziale delle tonsille (tonsillotomy). Pertanto, il numero di multinazionali ottenuti da campioni tonsillotomia (1 x ...

Tonsils are collections of incompletely encapsulated lymphoid tissues that lie under, and in contact with, the epithelium in the upper aero-digestive tract. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. The paired palatine tonsils together with the nasopharyngeal tonsil (adenoid), paired tubal tonsils and lingual tonsils constitute the so-called &#8220;Waldeyer&#180;s ring&#8221;. The latter is responsible for the initial contact between inhaled or ingested pathogens and the lymphoid tissues of the aerodigestive tract1,2. Indeed, numerous reports have shown that both bacterial and viral antigens can be detected in palatine tonsil tissue samples2-6.
The palatine tonsils are composed of dense lymphoid tissue covered by a stratified squamous non-keratinising epithelium. The tonsils have numerous crypts, epithelial invaginations, which penetrate the parenchyma increasing the surface area. Histologically, the palatine tonsils contain numerous lymphoid follicles with germinal centers, which are the sites for B cell maturation and differentiation (B-cell areas). Likewise, the palatine tonsils encompass T cells, which are mainly located in the extrafollicular regions (T-cell areas). In addition to the B and T cells, also various follicular dendritic cells can be detected in palatine tonsils1,2. 
Due to their anatomic location, the palatine tonsils are easily accessible by surgical interventions. For example, surgical removal of tonsils, known as tonsillectomy, is routinely carried out worldwide7. In children with tonsillar hyperplasia, a partial surgical removal of the tonsils (tonsillotomy) is sometimes used, causing less postoperative pain to the patients. Considering the accumulation of various pathogens in tonsils, surgically removed tonsils provide a unique opportunity to study the influence of viral and bacterial agents on tonsillar lymphocyte functions2,8. Furthermore it is possible to study if some pathogens prefer to reside in specific cell subpopulations9. In addition, as the tonsils are rich source of B lymphocytes, isolated tonsillar B lymphocytes can be efficiently used to study the activity of different B cell subpopulations10. However, as the palatine tonsils contain a mixture of cell types an efficient method to separate the different cell subpopulations is needed. 
Here, we describe a simple method for efficient and rapid isolation of tonsillar B and T cell populations from human palatine tonsils by using a magnetic-activated cell separation technique (Figure 1). The method described here is useful for scientists who want to assess the role of different infectious agents in human lymphoid organs such as palatine tonsils.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hanks balanced salt solution (HBSS)&#160;</td>
			<td>Gibco</td>
			<td>14175-053</td>
			<td>Contains 5% fetal bovine serum (FBS), 10 mM Glutamine,&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>&#160;0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix</td>
		</tr>
		<tr>
			<td>Freezing medium</td>
			<td></td>
			<td></td>
			<td>90% FBS and 10% DMSO</td>
		</tr>
		<tr>
			<td>MACS buffer</td>
			<td></td>
			<td></td>
			<td>PBS (pH 7.2), 0.5% BSA and 2mM EDTA</td>
		</tr>
		<tr>
			<td>PBSA&#160;</td>
			<td></td>
			<td></td>
			<td>PBS containing 0.2% BSA</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td></td>
			<td></td>
			<td>PBS containing 1% paraformaldehyde (PFA). Make fresh.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>PFA is suspected carcinogen. Wear gloves and goggles.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic mix</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>100-mm petridish</td>
			<td>Nunc</td>
			<td>172958</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting foreceps</td>
			<td>Fisher Scientific</td>
			<td>1381241</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight iris scissors&#160;</td>
			<td>Fisher Scientific</td>
			<td>12912055</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable scalpels</td>
			<td>Swann-Morton</td>
			<td>REF 0501</td>
			<td></td>
		</tr>
		<tr>
			<td>100&#956;m plastic cell strainer</td>
			<td>Corning Life Sciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m plastic cell strainers&#160;</td>
			<td>Corning Life Sciences</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>2-ml plastic syringe</td>
			<td>BD Biosciences&#160;</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>50-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62547254</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-speed centrifuge with fixed-angle or swinging-bucket rotor</td>
			<td colspan="2">Thermo Scientific Heraeus Megafuge 16R</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll&#8211;Hypaque</td>
			<td>Sigma-Aldrich</td>
			<td>F5415-50ML</td>
			<td>Ficoll solution</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Biological industries</td>
			<td>040071A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide (DMSO)</td>
			<td>SIGMA</td>
			<td colspan="2">D2650-5X5ML</td>
		</tr>
		<tr>
			<td>MACS MS columns&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS human CD3 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-881</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separator (Octo MACS)</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Merck Millipore</td>
			<td>1120180100</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>AnalaR NORMAPUR</td>
			<td>20302.293</td>
			<td></td>
		</tr>
		<tr>
			<td>CD2 conjugated to allophycocyanin (CD2-APC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>560642</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20 conjugated to fluorescein isothiocyanate (CD20-FITC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>556632</td>
			<td></td>
		</tr>
		<tr>
			<td>Human serum&#160;</td>
			<td>Rockland Immunochemicals</td>
			<td>D119-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F-530L</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>BD Falcon</td>
			<td>352003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotube</td>
			<td>SARSTEDT</td>
			<td>72379</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRII flowcytometer</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva 4.1 software</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Blood&#160;</td>
			<td>Macherey-Nagel</td>
			<td>740951.50</td>
			<td>DNA isolation kit</td>
		</tr>
		<tr>
			<td>TRIzol&#160; reagent</td>
			<td>Life technologies</td>
			<td>15596</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>The Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>0610030</td>
			<td>cell counter device</td>
		</tr>
		<tr>
			<td>GelRed</td>
			<td>Biotium</td>
			<td align="right">41003</td>
			<td>Nucleic acid gel stain&#160;</td>
		</tr>
	</tbody>
</table>

efficient isolation protocol for b and t lymphocytes from human palatine tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Una separazione efficace dei tonsillari MNC risultati in sottopopolazioni altamente purificate di linfociti B e T. Ciò è stato confermato mediante analisi FACS utilizzando anti-CD20 e anti-CD2 per rilevare B T linfociti popolazioni, rispettivamente (Figura 3A). Al contrario, una separazione efficiente di MNC risultati in frazioni cellulari che sono una miscela di cellule di entrambe le sottopopolazioni di linfociti B e T (Figura 3B). Il linfociti T <strong...

Watch this Scientific Journal Video about Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils at JoVE.com

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Efficiente protocollo Isolamento da B e T linfociti umani da Palatine Tonsille

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

efficient-isolation-protocol-for-b-t-lymphocytes-from-human-palatine

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

Immunology and Infection

Unit 1

Upper Respiratory Tract Infections

SCIENTISTS IN ACTION

15.7K Views.  Uppsala Biomedical Center, Uppsala University. Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Video: Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento linfociti T umani, la cultura e l&#39;analisi delle migrazioni In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Ricerca

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 ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Isolamento dei linfociti da mouse mucosa del tratto genitale

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Isolamento di precursori delle cellule B di sottoinsiemi di sangue del cordone ombelicale

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Isolamento e differenziazione di Th17 Naïve linfociti T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Analisi rapida di aberrazioni cromosomiche in linfociti B mouse da PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Trasfezione altamente efficiente di umani THP-1 da macrofagi Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Un semplice e rapido protocollo non-enzimaticamente dissociare freschi tessuti umani per l&#39;analisi di linfociti infiltranti

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Generazione di ricombinante umano IgG anticorpi monoclonali da immortalizzate cellule ordinati B

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

La differenziazione in vitro di cellule CD4 + umana FOXP3 + indotta da T regolatorie (iTregs) da cellule T CD4 Naïve utilizzando un protocollo TGF-β contenenti