Ringraziamo il Alvin J. Siteman Cancer Center della Washington University School of Medicine e Barnes-Jewish Hospital di St. Louis, MO, per l&#39;utilizzo del core ad alta velocità cell sorter, che ha fornito apparecchiature citometria a flusso e servizi di smistamento. Questo lavoro è stato sostenuto dal National Institutes of Borse Salute R01 HL082549 e K02 HL084179 (NRY). Il Siteman Cancer Center è sostenuto in parte da un supporto NCI Cancer Center di Grant P30 CA91842.

1. PREPARAZIONE DEI REAGENTI <ol><li> Preparare sterili soluzioni stock 1000x di Fms legati tirosin-chinasi 3 (FLT-3) ligando, granulociti / macrofagi colony-stimulating factor (GM-CSF), fattore della cellula staminale (SCF), trombopoietina (TPO), interleuchina (IL) -3 e IL-6 in base alle istruzioni del produttore. Preparare sterile soluzione madre Retronectin (1mg/ml) anche in base alle istruzioni del produttore. Dividere queste soluzioni madri in piccole aliquote e conservare a -20 ° C per evitare il ripetersi di congelamento e scongelamento. </li><li> Preparare il 2% FBS in IMDM. </li><li> Preparare terreno completo IMDM con concentrazioni finale del 20% FBS, 2 mM glutammina, 100 unità / ml di penicillina / streptomicina (PS). Aggiungere le seguenti citochine appena prima dell&#39;uso: 100 ng / ml FLT-3 ligando, 20 ng / ml GM-CSF, 100 ng / ml SCF, 100 ng / ml TPO, 50 ng / ml IL-3, e 100 ng / ml IL-6. </li><li> Preparare 1% BSA in HBSS (Ca e Mg-free, senza rosso fenolo) e 2% di BSA in D-PBS (Ca e Mg-free). Sterilizzare le soluzioni, passando attraverso i filtri siringa 0,22 micron e conservare a 4 ° C. </li><li> Preparare 0,02% EDTA in HBSS e conservare a 4 ° C. </li><li> Preparare GALV-pseudotyped retrovirus codifica del DNA di interesse e conservare in aliquote a -80 ° C. Questa procedura non rientra nell&#39;ambito di questo articolo, ma è descritto altrove (1-3). </li></ol> Per la colorazione Giemsa <ol start="7"><li> Appena prima dell&#39;uso, filtrare l&#39;Wright / Giemsa soluzione macchia due volte a 1 micron carta filtro in una bottiglia di vetro pulito. </li><li> Immediatamente prima dell&#39;uso, preparare Wright / Giemsa tampone pH 6,4, mescolando 50 ml del filtrato Wright / Giemsa soluzione macchia e 200 ml di soluzione tampone fosfato, pH 6,4 - Giordano formula. </li><li> Preparare una soluzione tampone fosfato, pH 6.0 sciogliendo 27,3 g di KH 2 PO 4 e 4,62 g di Na 2 HPO 4 in 3,5 LH 2 O. </li><li> Preparare metanolo al 50% in H 2 O. </li></ol> 2. PROCEDURA Scongelamento e pre-attivazione delle cellule CD34 + <ol><li> Scongelare una fiala di cellule CD34 + congelato rapidamente a 37 ° C agitando delicatamente fino a quando un ultimo cristallo di ghiaccio piccolo a sinistra e trasferire la sospensione cellulare (1 ml) in una provetta da 50 ml. Delicatamente risciacquare le celle rimanenti dalla fiala con 1 ml di temperatura ambiente 2% FBS / IMDM e aggiungerlo goccia a goccia per il tubo da 50 ml, mentre agitando delicatamente. Attendere 3 minuti. Poi, lentamente, aggiungere 2 ml del 2% FBS / IMDM, mescolando delicatamente, e attendere 3 minuti. Ripetere la procedura con l&#39;aggiunta di 2% FBS / IMDM che è lo stesso volume come la sospensione cellulare diluita ad intervalli di 3 minuti, mescolando delicatamente tra le aggiunte, fino al volume finale raggiunge i 32 ml. </li><li> Centrifugare a 250 xg per 10 minuti a temperatura ambiente e rimuovere il surnatante lasciando circa 0,5 ml. </li><li> Lavare il pellet con la sospensione in 20 ml al 2% FBS / IMDM e la centrifugazione a 200g per 8 minuti a temperatura ambiente. </li><li> Rimuovere il supernatante e sospendere le cellule in terreno IMDM completo a circa 0,5 x 10 6 cellule / ml. Prendere 10 ml di sospensione cellulare in un mix microtubo, con lo stesso volume di soluzione di Blu tripano (0,4%) e contare con un emocitometro. </li><li> Diluire le cellule a 0,1-0,15 X 10 6 cellule / ml con l&#39;aggiunta di terreno completo IMDM integrato con citochine (vedi 1.3) </li><li> Coltivare le cellule in una umidificata a 37 ° C incubatore con 5% di CO 2 per 2 giorni. </li></ol> Trasduzione retrovirale da virus pre-caricamento <ol start="7"><li> Il giorno della trasduzione, contare le celle prima di iniziare virus precarico per determinare il numero di pozzi da preparare. </li><li> Diluire la soluzione Retronectin magazzino a 25 mg / ml e aggiungere 400 microlitri di ciascun pozzetto di una 24-e non-trattati piastra di coltura. Incubare per 2 ore a temperatura ambiente nella cappa a flusso laminare biosicurezza di pre-coat. </li><li> Rimuovere la soluzione Retronectin e aggiungere 400 ml di BSA al 2% in D-PBS in ciascun pozzetto. Incubare per 30 minuti a temperatura ambiente. </li><li> Scongelare soluzioni madri virus e tenere in ghiaccio. Rimuovere la soluzione di BSA, aggiungere 0,5 ml di preparazione del virus in ciascun pozzetto e centrifugare a 2200 xg a 4 ° C per 15 minuti. </li><li> Rimuovere la soluzione di virus da pozzi e ripetere il caricamento del virus descritto in 2,10 altre tre volte. </li><li> Lavare ogni pozzetto con acqua fredda 0,5 ml HBSS (+ Ca e Mg) o IMDM. </li><li> Raccogliere le pre-attivato cellule CD34 + per centrifugazione a 200 xg per 8 minuti a temperatura ambiente. </li><li> Risospendere le cellule a 0,1-0,15 x 10 6 cellule per 1,5 ml in media IMDM completa di citochine. </li><li> Aggiungere 1,5 ml di sospensione cellulare in ciascun pozzetto e incubare umidificata in un incubatore a 37 ° C con 5% di CO 2 per 2 giorni. </li></ol> L&#39;ordinamento delle cellule e la raccolta di cellule trasdotte <ol start="16"><li> Delicatamente ma accuratamente sospendere le cellule del virus sopra-caricata piastras e filtrare attraverso un filtro di 50 micron CellTrics cellule in una provetta conica. Prendere 10 ml di sospensione cellulare in un mix microtubo, con un uguale volume di soluzione di Blu tripano e contare con un emocitometro. Lavare ogni pozzetto con 0,8 ml di freddo 0,02% EDTA / HBSS (Ca e Mg-free senza rosso fenolo) e aggiungere al tubo stessa collezione attraverso un filtro di 50 micron cellule CellTrics. </li><li> Centrifugare le cellule a 200g per 8 minuti a 4 ° C, e lavare il pellet con il freddo HBSS (Ca e Mg-free senza rosso fenolo) per centrifugazione a 200 xg per 8 minuti a 4 ° C. </li><li> Risospendere il pellet in 1% BSA / HBSS (Ca e Mg-free senza fenolo rosso) ad una concentrazione di 15 X 10 6 / ml o un volume minimo di 0,250 ml per l&#39;ordinamento delle cellule. Mantenere le cellule in ghiaccio nel buio. Preparare una provetta contenente 20% FBS freddo / IMDM / Glutammina / PS medio per la raccolta delle cellule ordinati. L&#39;ordinamento viene eseguito presso il Nucleo sorter ad alta velocità cella del J. Alvin Siteman Cancer Center della Washington University School of Medicine con una alta velocità MoFlo selezionatore (Dako, Glostrup, Danimarca). </li></ol> CFC test <ol start="19"><li> Scongelare il numero richiesto di 3 ml di mezzi di dosaggio CFC. Energicamente per mescolare e lasciare le provette in posizione almeno 5 minuti per consentire eventuali bolle presenti a salire in superficie prima di aggiungere le cellule. </li><li> Per ottenere la concentrazione di cellule per ogni campione ordinati, dividere il numero di cellulare fornito dalla struttura ordinamento in base al volume approssimativo sospensione cellulare. Prendere 3.000 virus trasdotte, le cellule ordinate in un microtubo sterile contenente fredda 2% FBS / IMDM. Pre-regolare il volume del 2% FBS / IMDM modo che il volume finale della sospensione sarà di circa 0,3 ml. </li><li> Sospendere le cellule e trasferire l&#39;intero 0,3 ml di sospensione cellulare ad una aliquota 3 ml di Methocult GF + H4435, che consiste di 1% di metilcellulosa, 30% FBS, 1% di BSA, 10 -4 M 2-mercaptoetanolo, 2 mM L-glutammina, 50 ng / ml SCF, 20 ng / ml GM-CSF 20 ng / ml di IL-3, 20 ng / ml di IL-6, 20 ng / ml G-CSF, e 3 unità / ml di eritropoietina in IMDM., Umano metilcellulosa Arricchito Media (R &amp; D Systems), che consiste di 1,3% di metilcellulosa, 25% FBS, 1% di BSA, 5 x 10 -5 M 2-mercaptoetanolo, 2 mM L-glutammina, 50 ng / ml SCF, 20 ng / ml di GM-CSF, 20 ng / ml di IL-3, 20 ng / ml di IL-6, 20 ng / ml G-CSF, e 3 unità / ml in IMDM eritropoietina può anche essere usato. </li><li> Energicamente per rendere il composto ascesa e la caduta completamente 3-4 volte. Lasciate che il tubo di stare fermo per 3 min. </li><li> Collegare un calibro 16 ago smussato-end di una siringa da 3 ml e redigere 2,2 ml. Non redigere le bolle di grandi dimensioni; espellerli all&#39;inizio spingendo fuori un paio di volte. Spingere fuori 1,1 ml ogni 30 millimetri in due non trattati piatto e distribuite il composto in modo uniforme mediante rotazione. </li><li> Luogo duplicare le piastre in un piatto 100 millimetri con un piatto di acqua contenente 3 ml di acqua sterile. Cultura per 14 - 17 giorni. </li><li> Caratterizzare e punteggio delle colonie in base alla loro morfologia con un microscopio rovesciato con ingrandimento 40x in un piatto di cultura contrassegnati con una griglia di punteggio. Ai fini del nostro test, le colonie sono classificati in 3 categorie: eritroide pura, mielomonocitica, e mista. Vedere le istruzioni del produttore per ulteriore sottoclassificazione colonia. </li><li> L&#39;intera piastra dosaggio CFC possono essere acquisiti senza ingrandimento usando un normale scanner a 600 dpi, e di bassa potenza (40x) microfotografie delle colonie possono essere scattate con un microscopio rovesciato dotato di una telecamera a colori. </li><li> Per ulteriori analisi di differenziazione e proliferazione, le cellule dalla piastra intero saggio CFC vengono recuperati con la sospensione in diversi volumi della temperatura ambiente 2% FBS / IMDM. Dopo centrifugazione a 300 xg per 10 minuti a 4 ° C, le cellule sono risospese in 2% FBS / IMDM, contati, e sia macchiato con anticorpi per citometria a flusso o trasferiti alle diapositive utilizzando una centrifuga Cytospin per la colorazione Giemsa. </li></ol> Anticorpi colorazione e citometria a flusso <ol start="28"><li> Cellule Centrifugare a 200 xg per 8 minuti a 4 ° C e risospendere a freddo nel siero del 50% del mouse normale in HBSS (Ca e Mg-free senza rosso fenolo). </li><li> Luogo di circa 5 x 10 5 cellule in 80 microlitri per tubo (12 x 75 mm a fondo rotondo tubi) e tenere su ghiaccio. Aggiungi miscela di anticorpi (circa 20 microlitri miscela o soluzione di controllo negativo per non-anticorpo controlli) e mescolare leggermente toccando i tubi. Preparare tubi di controllo della calibrazione, senza o con un anticorpo anti-CD45 anticorpi per ogni fluorocromo. Tubi posto sul ghiaccio al buio e incubare per 20 minuti. Mescolare picchiettando leggermente provette dopo i primi 10 min. Dopo l&#39;incubazione 20 min, riempire i tubi con HBSS freddo e centrifugare a 200 xg per 8 minuti a 4 ° C. Risospendere il pellet in freddo paraformaldeide 0,5% / HBSS. Le seguenti miscele di anticorpi sono generalmente utilizzati per la nostra analisi. Per la differenziazione mieloide: CD11b (ficoeritrina coniugati clone ICRF44) / CD33 (allophycocyanin coniugato clone WM53) / CD45 (ficoeritrina-Cy7-congjugated clone HI30). Per la differenziazione eritroide: CD71 (ficoeritrina-coniugato clone M-A712) / CD235a (allophycocyanin coniugata con clone GA-R2) / CD45 (ficoeritrina-Cy7-congjugated clone HI30). </li><li> Citometria a flusso viene eseguita al core ad alta velocità Sorter cella del J. Alvin Siteman Cancer Center della Washington University School of Medicine su un citofluorimetro FACScan aggiornato a 5 colori e due laser (BD Biosciences) e analizzati utilizzando CellQuest (BD Biosciences) o FlowJO v7.2.4 (Albero Star, Inc., Ashland, OR, USA) del software. </li></ol> Colorazione Giemsa <ol start="31"><li> Circa 30.000 cellule recuperate dai piatti dosaggio CFC sopra descritte sono sospesi in 0,3 ml 2% FBS / IMDM e trasferito a una diapositiva con una centrifuga Cytospin a 1.000 rpm per 10 minuti a temperatura ambiente. </li><li> Impostare nove navi colorazione contenenti soluzioni nel seguente ordine. <ol><li> Metanolo assoluto </li><li> Wright / Giemsa soluzione stock </li><li> Wright / Giemsa tampone, pH 6,4 </li><li> 50% di metanolo in H 2 O </li><li> H 2 O </li><li> H 2 O </li><li> Tampone fosfato, pH 6,0 </li><li> H 2 O </li><li> H 2 O </li></ol></li><li> Porre i vetrini in un vettore di diapositive e tuffo in metanolo assoluto (# 1) per 2 minuti e asciugare fuori metanolo in eccesso. </li><li> Immediatamente, immergere il vettore diapositiva Wright / Giemsa soluzione madre (# 2) per 5 min. </li><li> Trasferire il vettore in Wright / Giemsa, pH 6.4 (# 3) e incubare per 10 min. </li><li> Immergere il vettore due volte in metanolo al 50% (# 4), 10 volte in H 2 O (# 5), un altro 10 volte in H 2 O (# 6), e poi 5 volte in tampone fosfato, pH 6.0 (# 7) . </li><li> Trasferire il vettore in H 2 O (# 8) e incubare per 2 minuti. Ripetere il lavaggio in H 2 O (# 9). </li><li> Lasciare le diapositive asciugare completamente nel supporto. Rimuovere ogni diapositiva e pulire la parte posteriore del vetrino con metanolo-imbevuto Kimwipes per rimuovere le macchie. Mettere una goccia di Cytoseal 60 e un vetro di copertura per sigillare. </li><li> Un 500-conta delle cellule differenziale è eseguita per ogni Giemsa-macchiate presentazione utilizzando un microscopio Olympus BX51. Le cellule si dividono in cinque categorie: le cellule primitive comprendono blasti e di promielociti; cellule mieloidi intermedie comprendono mielociti e metamielociti; cellule mieloidi mature comprendono bande, neutrofili segmentati, monociti e macrofagi, cellule eritroidi intermedia includono cellule con hemoglobinization intermedi; e maturare cellule eritroidi sono cellule con hemoglobinization pieno. Microfotografie sono scattate con una fotocamera Olympus DP71 con un obiettivo 60x olio. </li></ol> 3,0) risultati rappresentativi: <ol><li> Dosaggio CFC: Alcune delle popolazione di cellule ingresso sarà proliferano, si differenziano e colonie modulo a semi-solida media durante il 14-17 giorni di incubazione. In questo esperimento, è evidente che l&#39;espressione del NUP98 HOXA9-oncogene causato la formazione di importanti rosso (colonie eritroidi) (Figura 1) (3). </li><li> La colorazione degli anticorpi e Citometria a Flusso: immunofenotipizzazione citometria a flusso delle cellule raccolte da piastre test CFC fornisce informazioni sullo stato di differenziazione della popolazione cellulare. Con l&#39;utilizzo combinato di anticorpi contro marcatori di differenziazione certo, la discendenza e il grado di maturazione delle cellule può essere valutata. In questo caso CD235a è stato utilizzato per identificare le cellule eritroidi. Come mostrato nella Figura 2A, NUP98-HOXA9 causato un aumento della percentuale di CD235a + cellule eritroidi, ma la luminosità di espressione CD235a da queste cellule è stato ridotto rispetto ai controlli, indicando l&#39;inibizione della maturazione eritroide. In Figura 2B, le cellule mieloidi sono identificati in virtù della loro positività CD33. NUP98-HOXA9 causato una diminuzione del numero di cellule CD33 +, con una diminuzione della luminosità di espressione CD11b, coerente con l&#39;inibizione della maturazione mieloide (3). Così i risultati mostrano che la citometria di flusso NUP98-HOXA9 causato un iperplasia eritroide con l&#39;inibizione di maturazione sia mieloide ed eritroide. </li><li> Colorazione Giemsa: esame morfologico delle cellule raccolte da piastre test CFC fornisce informazioni sul lignaggio e il grado di maturazione della popolazione cellulare. Le conclusioni ottenute morfologicamente possono differire da quelli ottenuti da studi di citometria a flusso, e quindi questi metodi sono complementari gli uni agli altri (3). L&#39;esperimento viene ripetuto almeno 3 volte, e ogni volta che un 500-celle conta differenziale è eseguita a distinguere 5 categorie di cellule, come descritto sopra. Esempi di queste cellule sono segnalate nella Figura 3. I risultati sono tabulati e analizzati al fine di determinare se vi sono differenze statisticamente significative tra l&#39;oncogene-trasdotte campione e il controllo. In questo caso, i risultati hanno mostrato che NUP98-HOXA9 ha causato un aumento complessivo del numeros delle cellule, con iperplasia eritroide e l&#39;inibizione di entrambe le eritroide e maturazione mieloide (3) (non mostrato). </li></ol> FIGURA LEGENDS <img src="/files/ftp_upload/2195/2195fig1.jpg" alt="Figura 1" /> Figura 1: Gli effetti del NUP98-HOXA9 sulla morfologia CFC umano. Primarie umane cellule CD34 + sono state trasdotte con retrovirally sia MSCV-IRES-GFP controllo vettoriale o vettoriale che esprime NUP98-HOXA9, e le cellule sono state ordinate per la positività GFP. Un migliaio di cellule sono state seminate in ognuna delle due piastre duplicati per dosaggio CFC e l&#39;esperimento è stato ripetuto 3 4 volte indipendenti. Piastre rappresentante senza ingrandimento (a sinistra) e microfotografie a basso consumo di rappresentante colonie eritroidi (a destra) sono indicati (3). <img src="/files/ftp_upload/2195/2195fig2.jpg" alt="Figura 2" /> Figura 2: La citometria a flusso mostra interruzione umano primario differenziazione delle cellule CD34 + da NUP98-HOXA9. (A) citometria a flusso per il differenziamento eritroide: Cellule dalle piastre CFC (vedi Figura 1) sono state raccolte e colorate con anticorpi CD45 e CD235a. Il CD235a + porta era tracciati su un istogramma (pannello di destra) per mostrare l&#39;espressione di CD235a rispetto alle cellule di controllo (B) citometria a flusso per la differenziazione mieloide: Cellule dalle piastre CFC sono state raccolte e colorati con CD45 e CD33. Il CD33 + cancello è stato tracciato su un istogramma per mostrare l&#39;espressione CD11b rispetto al controllo (pannello di destra). Le percentuali di cellule che rientrano ogni porta sono mostrati (3). <img src="/files/ftp_upload/2195/2195fig3.jpg" alt="Figura 3" /> Figura 3: morfologia cellulare mostra interruzione di differenziazione da parte NUP98-HOXA9 rispetto al controllo vettoriale. Strisci Cytospin sono stati preparati dai piatti CFC e colorati con Giemsa. Microfotografie sono state prese dai campi di rappresentanza con un obiettivo 60x olio. B: esplosione; MM: mieloide maturo, IM: mieloide intermedio, ME: maturo eritroide, IE: intermedio eritroidi (3).

<ol>
	<li>Takeda, A., Goolsby, C., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NUP98-HOXA9+induces+long-term+proliferation+and+blocks+differentiation+of+primary+human+CD34++hematopoietic+cells.">NUP98-HOXA9 induces long-term proliferation and blocks differentiation of primary human CD34+ hematopoietic cells.</a> Cancer Res. , 66-6628 (2006).</li><li>Yassin, E. R., Abdul-Nabi, A. M., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+NUP98-DDX10+oncogene+on+primary+human+CD34++cells:+role+of+a+conserved+helicase+motif.">Effects of the NUP98-DDX10 oncogene on primary human CD34+ cells: role of a conserved helicase motif.</a> Leukemia. , (2010).</li><li>Yassin, E. R., Sarma, N. J., Abdul-Nabi, A. M., Dombrowski, J., Han, Y., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+transformation+of+primary+human+hematopoietic+cells+by+the+oncogene+NUP98-HOXA9.">Dissection of the transformation of primary human hematopoietic cells by the oncogene NUP98-HOXA9.</a> PLoS One. 4, e6719-e6719 (2009).</li><li>Nissen-Druey, C., Tichelli, A., Meyer-Monard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hematopoietic+colonies+in+health+and+disease.">Human hematopoietic colonies in health and disease.</a> Acta Haematol. 113, 5-96 (2005).</li><li>Pereira, C., Clarke, E., Damen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+colony-forming+cell+assays.">Hematopoietic colony-forming cell assays.</a> Methods Mol Biol. 407, 177-208 (2007).</li><li>Coulombel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+hematopoietic+stem/progenitor+cells:+strength+and+drawbacks+of+functional+assays.">Identification of hematopoietic stem/progenitor cells: strength and drawbacks of functional assays.</a> Oncogene. 23, 7210-7222 (2004).</li><li>Hogge, D. E., Lansdorp, P. M., Reid, D., Gerhard, B., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection,+maintenance,+and+differentiation+of+primitive+human+hematopoietic+cells+in+cultures+containing+murine+fibroblasts+engineered+to+produce+human+steel+factor,+interleukin-3,+and+granulocyte+colony-stimulating+factor.">Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor.</a> Blood. 88, 3765-3773 (1996).</li></ol>

Nessun conflitto di interessi dichiarati.

Il dosaggio CFC è stato ampiamente utilizzato per determinare la proliferazione e la differenziazione dei modelli di progenitori emopoietici e per studiare gli effetti di oncogeni (4, 5). Ha il vantaggio rispetto a colture liquide di essere un saggio clonale, tanto che le colonie rappresentano la progenie di un solo progenitore e può essere rimossa singolarmente per ulteriori analisi. La limitazione del dosaggio CFC è che non è adeguata per la rilevazione dei progenitori più immaturi o cellule staminali ematopoieti...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>IMDM</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>12440</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>06150</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin (PS)</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>FLT-3 ligand</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-19</td>
<td>&nbsp;</td>
<tr>
<td>GM-CSF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-03</td>
<td>&nbsp;</td>
<tr>
<td>SCF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-07</td>
<td>&nbsp;</td>
<tr>
<td>TPO</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-18</td>
<td>&nbsp;</td>
<tr>
<td>IL3</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-03</td>
<td>&nbsp;</td>
<tr>
<td>IL6</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-06</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma- Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP118</td>
<td>&nbsp;</td>
<tr>
<td>Retronectin</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>T100A</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14175</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14200</td>
<td>&nbsp;</td>
<tr>
<td>Trypan Blue solution (0.4%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8154</td>
<td>&nbsp;</td>
<tr>
<td>Methocult GF+ H4435</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>04445</td>
<td>&nbsp;</td>
<tr>
<td>Human Methylcellulose Enriched Media</td>
<td>&nbsp;</td>
<td>R &amp; D Systems</td>
<td>HSC005</td>
<td>&nbsp;</td>
<tr>
<td>Wright/ Giemsa stain</td>
<td>&nbsp;</td>
<td>Harleco</td>
<td>64571</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffer Solution, pH 6.4 - Giordano formula</td>
<td>&nbsp;</td>
<td>Ricca Chemical Company</td>
<td>1450</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A412-4</td>
<td>&nbsp;</td>
<tr>
<td>Cytoseal 60</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>8310</td>
<td>&nbsp;</td>
<tr>
<td>Normal Mouse Serum </td>
<td>&nbsp;</td>
<td>Rockland Immunochemicals</td>
<td>D208</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD11b phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555388</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD33 allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551378</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD71 phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555537</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD235a allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551336</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>24-well non-treated tissue culture plates</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-1147</td>
<td>&nbsp;</td>
<tr>
<td>30 mm non-treated dish</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27150 </td>
<td>&nbsp;</td>
<tr>
<td>100 mm tissue culture dish </td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27500</td>
<td>&nbsp;</td>
<tr>
<td>15 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2097</td>
<td>&nbsp;</td>
<tr>
<td>50 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2070</td>
<td>&nbsp;</td>
<tr>
<td>Syringes 3 ml </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28240</td>
<td>&nbsp;</td>
<tr>
<td>16 gauge blunt-end, 1&frac12; inch needle </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28110</td>
<td>&nbsp;</td>
<tr>
<td>50 &mu;m CellTrics cell filter</td>
<td>&nbsp;</td>
<td>Partec</td>
<td>04-004-2327</td>
<td>&nbsp;</td>
<tr>
<td>Hemocytometer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>0267110</td>
<td>&nbsp;</td>
<tr>
<td>TPX sample chambers</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>A78710018</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Superfrost/Plus Microscope Slides, Precleaned</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-550-15</td>
<td>&nbsp;</td>
<tr>
<td>Shandon filter cards</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>5991022</td>
<td>&nbsp;</td>
<tr>
<td>Shandon cytospin slide holder</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>59920063</td>
<td>&nbsp;</td>
<tr>
<td>Shandon Complete Staining Assembly 100</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>100</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>Kimberly-Clark Corporation</td>
<td>34155</td>
<td>&nbsp;</td>
<tr>
<td>1 &mu;m filter paper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28307-134</td>
<td>&nbsp;</td>
<tr>
<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>Diaphot</td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>DS-F11</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX51 </td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>DP71</td>
<td>&nbsp;</td>
<tr>
<td>Scanner</td>
<td>&nbsp;</td>
<td>Microtek</td>
<td>Scanmaker 4</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-812</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture incubator</td>
<td>&nbsp;</td>
<td>Sanyo</td>
<td>MCO-18AIC</td>
<td>&nbsp;</td>
<tr>
<td>Cytospin</td>
<td>&nbsp;</td>
<td>Shandon</td>
<td>Cytospin 2</td>
<td>&nbsp;</td>
<tr>
<td>Bench-top centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5810-R</td>
<td>&nbsp;</td>
<tr>
<td>Water purification system</td>
<td>&nbsp;</td>
<td>Barnstead</td>
<td>Nanopure-Diamond</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

colony forming cell (cfc) assay for human hematopoietic cells

Umane ematopoietiche staminali / progenitrici sono solitamente ottenute da midollo osseo, sangue del cordone ombelicale, o dal sangue periferico e sono utilizzati per lo studio ematopoiesi e leucemogenesi. Hanno la capacità di differenziarsi in linee linfoidi e mieloidi. La colonia di cellule che formano (CFC) test è usato per studiare la proliferazione e la differenziazione modello di progenitori emopoietici con la loro capacità di formare colonie in un mezzo semisolido. Il numero e la morfologia delle colonie formate da un numero fisso di celle di input fornire informazioni preliminari circa la capacità dei progenitori di differenziarsi e proliferare. Le cellule possono essere raccolte da singole colonie o da tutta la piastra per valutare ulteriormente il loro numero e gli stati di differenziazione in citometria a flusso e la valutazione morfologica di Giemsa vetrini colorati. Questo test è utile per valutare la differenziazione mieloide, ma non linfoidi. Il mieloide termine in questo contesto è usato nel suo senso più ampio fino a comprendere lignaggi granulocitica, monociti, eritroide e megacariociti. Abbiamo usato questo test per valutare gli effetti di oncogeni sulla differenziazione delle primarie umane cellule CD34 + derivate dal sangue periferico. Per questo scopo sono le cellule trasdotte con entrambi costruire controllo retrovirali o un costrutto che esprime l&#39;oncogene di interesse, in questo caso NUP98-HOXA9. Ci avvaliamo di un vettore retrovirale comunemente usato, MSCV-IRES-GFP, che esprime un mRNA bicistronic che produce il gene di interesse e di un marcatore GFP. Le cellule sono pre-attivate crescendo in presenza di citochine per due giorni prima della trasduzione retrovirale. Dopo altri due giorni, le cellule GFP + sono isolate dalla fluorescenza-attivato cell sorting (FACS) e mescolato con un metilcellulosa mezzo contenente semisolido integrato con citochine e incubate fino colonie appaiono sulla superficie, in genere 14 giorni. Il numero e la morfologia delle colonie sono documentati. Le cellule vengono poi rimossi dalle piastre, lavato, contati, e sottoposti a citometria a flusso e l&#39;esame morfologico. Citometria a flusso con anticorpi specifici marcatori di superficie della cellula espressi durante emopoiesi fornisce informazioni sulla genealogia e lo stadio di maturazione. Studi morfologici delle singole cellule al microscopio dopo la colorazione di Wright-Giemsa fornire ulteriori informazioni in materia di genealogia e di maturazione. Confronto di cellule trasdotte con controllo vettoriale vuoto a quelli trasdotte con un oncogene rivela gli effetti del oncogene sulla differenziazione ematopoietiche.

Watch this Scientific Journal Video about Colony Forming Cell CFC Assay for Human Hematopoietic Cells at JoVE.com

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

La colonia di cellule che formano (CFC) test è un test in vitro in cui progenitori emopoietici formare colonie in terreno semisolido. Una combinazione di morfologia delle colonie, la morfologia delle cellule, e citometria a flusso sono utilizzati per valutare la capacità dei progenitori di proliferare e differenziarsi lungo le varie linee ematopoietiche.

Formanti colonia cellulare (CFC) Analisi per cellule emopoietiche

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

colony-forming-cell-cfc-assay-for-human-hematopoietic-cells

Research

JoVE Journal

Immunology and Infection

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

35.5K Views.  Washington University School of Medicine. La colonia di cellule che formano (CFC) test è un test in vitro in cui progenitori emopoietici formare colonie in terreno semisolido. Una combinazione di morfologia delle colonie, la morfologia delle cellule, e citometria a flusso sono utilizzati per valutare la capacità dei progenitori di proliferare e differenziarsi lungo le varie linee ematopoietiche.

Video: Colony Forming Cell CFC Assay for Human Hematopoietic Cells

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Generazione di Multivirus-cellule T specifiche per prevenire / curare infezioni virali dopo trapianto allogenico di cellule staminali ematopoietiche

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Ricerca

Immunologia e infezioni

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

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

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

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

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

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

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&amp;#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

L&#39;uso di interferone-γ Enzyme-linked Assay Immunospot per caratterizzare Nuovi epitopi T-cellulari di Papillomavirus Umano

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac Transposon modifica del sistema di cellule primarie umane T

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

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 ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

L&#39;identificazione del DNA Mutazioni in depurata ematopoietiche cellule staminali / progenitrici

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

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

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

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

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

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

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

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Cultura dei macrofagi macrofagi colony-stimulating factor differenziata umana derivate da monociti

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Un'analisi di citotossicità di base di citometria a flusso per la valutazione dell'attività delle cellule NK umane

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Sviluppo e validazione di una singola molecola ultrasensibile di matrice digitale analisi enzima-collegata dell'immunosorbente per umana dell'interferone-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...