This work was supported by the key projects of Chinese Academy of Sciences (KFZD-SW-205), strategic biological resources technology support system of Chinese Academy of Sciences (CZBZX-1).

Questo studio è stato approvato dal Comitato Etico dell&#39;Istituto di Genetica e Biologia dello sviluppo, e il protocollo segue le linee guida istituzionali per il benessere umano. 1. Preparazione di EB Virus <ol><li> Il giorno 1, scongelare B95-8 cellule e di coltura in 6 mL completo RPMI 1640 medium, supplementato con 10% siero fetale bovino, 2 mM L-glutammina e antibiotici (100 mg / ml di streptomicina, 100 U / ml penicillina) in un T25 pallone di coltura. Mettere il pallone di coltura in 5% CO 2 incubatore a 37 ° C. </li><li> Il giorno 2, osservare la morfologia delle cellule al microscopio. Le cellule sono in buone condizioni quando sono luminose e rotondo. Aggiungere 10 ml completo di media RPMI 1640. </li><li> Il giorno 4, aggiungere 10 ml di fresco completo RPMI 1640, e poi trasferire in un pallone di coltura T75. </li><li> Il giorno 8, aggiungere 20 ml completo di media RPMI 1640. </li><li> Mantenere le cellule per 8 giorni senza l&#39;aggiunta di mezzo fresco, vale a dire, la fame. </li><li> Il giorno 16, osservare le celluleosservata al microscopio. Raccogliere le cellule in una provetta da 15 centrifuga, 12 ml / provetta. </li><li> Cryo-conservazione a -80 ° C per 1 ora, e poi rapidamente scongelare a 37 ° C. Ripetere 3 volte fino a quando le particelle virali vengono rilasciati dalle cellule. </li><li> Centrifugare a 240 xg per 10 min a temperatura ambiente. </li><li> Filtrare il surnatante attraverso un filtro di 0,22 micron, aliquota, 12 mL / tubo, e conservare a -80 ° C. </li></ol> 2. Trattamento di sangue intero con Globulo rosso Lysis Buffer <ol><li> Aggiungere 0,5 ml di sangue intero anticoagulato con EDTA · K 2 in una provetta da 15 ml. </li><li> Mettere 1 ml tampone di lisi dei globuli rossi (filtrati da 0,22 micron filtro e scongelati a temperatura ambiente prima dell&#39;uso) per il sangue intero e mescolare bene. Posizionare la miscela a temperatura ambiente per 4 min. Poi aggiungere 5 - 7 mL PBS 1x rapidamente alla reazione di arresto. </li><li> Centrifugare a 240 xg per 5 min a temperatura ambiente. I globuli bianchi precipitano sul fondo della provetta. </li><li> Eliminare il surnatante e lavare la precipitazione con l&#39;aggiunta di 7 ml 1640 medium di base. Centrifugare per 5 minuti a 240 xg a temperatura ambiente. Ripetere due volte. </li><li> Risospendere il pellet di cellule in 300 microlitri di media trasformazione (terreno RPMI 1640, contenente 20% di siero fetale bovino e 10 ug / ml fitoemoagglutinina (PHA)). </li></ol> 3. EBV infezione <ol><li> Aggiungere 60 ml di 20 mg / ml ciclosporina A (CsA) alla sospensione cellulare. </li><li> Rapidamente scongelare un&#39;aliquota di EBV a 37 ° C. Aggiungere 240 microlitri EBV alla sospensione cellulare. </li><li> Mescolare bene e mettere la sospensione cellulare ad una piastra a 96 pozzetti, 200 ml per pozzetto. Per evitare l&#39;evaporazione, aggiungere 200 microlitri di PBS 1x ad ogni bene intorno della piastra a 96 pozzetti. Porre la piastra in un incubatore CO 2 5% a 37 ° C. </li></ol> 4. Ampliamento e crioconservazione di B-LCL <ol><li> Modificare la metà del mezzo una volta a settimana per il primo mese dopo l&#39;infezione da EBV. Solo un laye spessorer di frammenti cellulari è visibile al microscopio al giorno 7 (figura 2). Dopo un mese di infezione EBV, gruppi di cellule linfoblastoidi sono visibili sotto uno spesso strato di frammenti cellulari al microscopio (Figura 2). </li><li> Cambiare la metà del mezzo ogni 3 giorni fino a quando i gruppi di cellule sono chiaramente visibili al microscopio (Figura 2). In genere, questo processo richiede da 10 a 20 giorni. </li><li> Dopo la miscelazione, pipettare le cellule e trasferire alla piastra 24 pozzetti. Raddoppiare il volume di terreno di coltura con completo RPMI 1640 mentre diventa giallo. Questo processo dipende dal tasso di crescita cellulare. </li><li> Successivamente, trasferire le cellule a pallone di coltura T25. Raddoppiare il volume del terreno di coltura come risulta giallo finché il volume del mezzo espande a 10 - 15 mL. </li><li> Cambiare media 24 ore prima del congelamento. sospensione cellulare Centrifugare per 5 min a 240 x g. Contare le cellule con un contatore di cellule e congelare ad una densità di 5 - 10 x 10 6 / ml in frSoluzione Ozen contenente 90% FBS e il 10% dimetilsolfossido (DMSO). Congelare a una velocità di 1 ° C per min a -80 ° C e poi trasferire direttamente in azoto liquido. </li></ol> 5. 3- (4,5-dimetiltiazol-2-il) -2,5-difenil-tetrazolio bromuro (MTT) Assay 17 <ol><li> Preparare una sospensione singola cellula B-LCL con il mezzo 1640 contenente il 10% di siero fetale bovino. Seed 3 x 10 4 cellule per pozzetto sulla piastra da 96 pozzetti, 100 ml per pozzetto. </li><li> Porre la piastra in 5% CO 2 incubatore a 37 ° C per 12 - 16 h. </li><li> Aggiungere MTT (5 mg / ml) per i pozzi in cui le cellule sono in crescita e tenere lontano dalla luce per 4 ore a 37 ° C. </li><li> piastra di cella Centrifugare per 10 min a 168 x g. </li><li> Rimuovere il surnatante e aggiungere 100 ml di DMSO per pozzetto. NOTA: Attenzione. Non toccare i cristalli di colorante aghiformi. </li><li> Agitare la piastra per 10 min a bassa velocità per sciogliere completamente i cristalli. </li><li> l&#39;assorbimento di misura a 570 nm perutilizzando uno spettrofotometro per micropiastre. </li></ol>

<ol>
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H., Quackenbush, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+lymphoblastoid+cell+lines+from+monozygotic+twins+discordant+in+severity+of+autism+reveals+differential+regulation+of+neurologically+relevant+genes.">Gene expression profiling of lymphoblastoid cell lines from monozygotic twins discordant in severity of autism reveals differential regulation of neurologically relevant genes.</a> BMC Genomics. 7, 118 (2006).</li><li>Toda, T., Sugimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Epstein-Barr+virus-transformed+B-lymphoblasts+and+the+proteome+database.">Proteome analysis of Epstein-Barr virus-transformed B-lymphoblasts and the proteome database.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 787 (1), 197-206 (2003).</li><li>Chiorazzi, N., Wasserman, R. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+the+rapid+establishment+of+Epstein-Barr+virus+immortalization+of+human+B+lymphocytes.">An efficient method for the rapid establishment of Epstein-Barr virus immortalization of human B lymphocytes.</a> Cell Prolif. 36 (4), 191-197 (2003).</li><li>Louie, L. G., King, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel-Approach+to+Establishing+Permanent+Lymphoblastoid+Cell-Lines+-+Epstein-Barr-Virus+Transformation+of+Cryopreserved+Lymphocytes.">A Novel-Approach to Establishing Permanent Lymphoblastoid Cell-Lines - Epstein-Barr-Virus Transformation of Cryopreserved Lymphocytes.</a> Am J Hum Genet. 48 (3), 637-638 (1991).</li><li>Tremblay, S., Khandjian, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+use+of+long-term+frozen+lymphocytes+for+the+establishment+of+lymphoblastoid+cell+lines.">Successful use of long-term frozen lymphocytes for the establishment of lymphoblastoid cell lines.</a> Clin Biochem. 31 (7), 555-556 (1998).</li><li>Reidy, J. A., Wheeler, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+age+and+Epstein-Barr+virus+transformation+of+cryopreserved+lymphocytes.">Sample age and Epstein-Barr virus transformation of cryopreserved lymphocytes.</a> In Vitro Cell Dev Biol. 28 (6), 383-384 (1992).</li><li>Amoli, M. M., Carthy, D., Platt, H., Ollier, W. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EBV+immortalization+of+human+B+lymphocytes+separated+from+small+volumes+of+cryo-preserved+whole+blood.">EBV immortalization of human B lymphocytes separated from small volumes of cryo-preserved whole blood.</a> Int J Epidemiol. 37, 41-45 (2008).</li><li>Lacelle, C., Wang, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+lymphoblastoid+cell+lines+from+frozen+blood+of+extremely+old+individuals.">Establishing lymphoblastoid cell lines from frozen blood of extremely old individuals.</a> Mech Ageing Dev. 123 (10), 1415-1418 (2002).</li><li>Tohda, H., Oikawa, A., Kudo, T., Tachibana, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+greatly+simplified+method+of+establishing+B-lymphoblastoid+cell+lines.">A greatly simplified method of establishing B-lymphoblastoid cell lines.</a> Cancer Res. 38 (10), 3560-3562 (1978).</li><li>Ventura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+Simple+Method+for+the+Epstein-Barr+Virus+Transformation+of+Lymphocytes+from+Members+of+Large+Families+of+Reunion+Island.">Use of a Simple Method for the Epstein-Barr Virus Transformation of Lymphocytes from Members of Large Families of Reunion Island.</a> Hum Hered. 38 (1), 36-43 (1988).</li><li>Chenevixtrench, G., Kerr, B., Walters, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoblastoid+Cell-Lines+from+Frozen+Whole-Blood+-+a+Quick+and+Economical+Safeguard+for+Linkage+Analysis.">Lymphoblastoid Cell-Lines from Frozen Whole-Blood - a Quick and Economical Safeguard for Linkage Analysis.</a> Am J Hum Genet. 46 (1), 635-636 (1990).</li><li>Hansen, M. 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E., Berg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-Examination+and+Further+Development+of+a+Precise+and+Rapid+Dye+Method+for+Measuring+Cell-Growth+Cell+Kill.">Re-Examination and Further Development of a Precise and Rapid Dye Method for Measuring Cell-Growth Cell Kill.</a> J Immunol Methods. 119 (2), 203-210 (1989).</li><li>Bogoslovsky, 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=Preservation+and+enumeration+of+endothelial+progenitor+and+endothelial+cells+from+peripheral+blood+for+clinical+trials.">Preservation and enumeration of endothelial progenitor and endothelial cells from peripheral blood for clinical trials.</a> Biomark Med. 9 (7), 625-637 (2015).</li><li>Ambach, A., Bonnekoh, B., Gollnick, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+flow+cytometric+immuno-staining+of+T-cell+perforin+is+preserved+using+diethylene+glycol+for+erythrocyte-lysis+but+lost+by+the+use+of+ammonium+chloride.">Routine flow cytometric immuno-staining of T-cell perforin is preserved using diethylene glycol for erythrocyte-lysis but lost by the use of ammonium chloride.</a> Exp Dermatol. 12 (6), 825-831 (2003).</li><li>Beck, J. 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The authors declare that they have no competing financial interests.

Riportiamo lo sviluppo di un nuovo metodo per immortalare cellule B umane da sangue intero mediante lisi dei globuli rossi, e la sua vitalità cellulare dei stabilita B-LCL è stata valutata mediante il saggio MTT. I risultati hanno mostrato che la vitalità cellulare di B-LCL stabilita con il metodo di lisi dei globuli rossi è molto superiore a quella del metodo di separazione gradiente di densità. Il principale vantaggio del metodo di lisi dei globuli rossi è che è semplice e richiede un piccolo volume (non più di 0,5 ml) di sangue per stabilire una linea cellulare permanente con un alto tasso di successo e alta vitalità cellulare. Solo è necessario un semplice passo lisi prima infezione da EBV. Senza precedente purificazione dei linfociti, la perdita di cellule e danni cellulari sono evitati. Un altro ostacolo del metodo di separazione di densità gradiente è emolisi. Elaborazione del sangue dopo la conservazione a lungo termine a temperature inadeguate e scuotendo durante il trasporto può causare emolisi, which influenzerà notevolmente l&#39;isolamento dei linfociti. Altri studi hanno dimostrato che l&#39;uso di globuli rossi (RBC) lisi portato a un numero maggiore di vitale PBMC che l&#39;uso del metodo di separazione gradiente di densità 18 e linfociti sottotipi non sono stati influenzati sostanzialmente 19. Rispetto al metodo di separazione gradiente di densità, che è costosa e ingombrante, il metodo di lisi dei globuli rossi è molto più facile, soprattutto per il trattamento di un gran numero di campioni clinici e più pratico per principianti. Nello studio, abbiamo immortalato 24 campioni con un tasso di successo del 100% utilizzando il metodo di lisi dei globuli rossi. Rispetto al 90-94% del tasso di successo riportato da linfociti crioconservati e linfociti appena isolate 20 con 2-2,5 ml di sangue, questo tasso di successo è molto più alto. Nel frattempo, abbiamo anche trasformato fresco (0,5 ml) o congelati di sangue (anche 1 ml), come descritto nei riferimenti 14,15 senza separazione, e l&#39;efficienzecy di trasformazione è molto più basso di quello riportato 16 (dati non mostrati). L&#39;attuale strategia non solo migliora l&#39;efficienza immortalizzazione in modo significativo, ma riduce anche il volume di sangue necessario per stabilire B-LCL. Tecnologie attuali per EBV trasformazione uso relativamente grandi volumi di sangue periferico, a circa 2 - 2,5 ml di sangue da 20 a isolare i linfociti, mentre altri richiedono 10 ml o più 10,21. Ciò contrasta con 0,5 ml di sangue intero necessarie per ottenere una linea cellulare immortale con il metodo qui descritto. Questo metodo potrebbe essere modificato per immortalare con successo raccolto dal dito del sangue punge 12, che potrebbe notevolmente facilitare preservare le risorse genetiche di pazienti pediatrici e anziani. Inoltre, la vitalità cellulare di B-LCL stabilita con il metodo di lisi dei globuli rossi sono superiore a quella del metodo di separazione gradiente di densità. La cella l sangue rossoYsis metodo evita la forza meccanica centrifuga nel processo di centrifugazione in gradiente di densità, che provoca danni non riparabili alle cellule. Si suggerisce che il metodo di lisi dei globuli rossi è più adatto per studi genetici e funzionali, che richiede un gran numero di cellule più rapidamente possibile. Il nostro metodo si riduce il tempo dalla cultura iniziazione alla crioconservazione, che farà risparmiare risorse e ridurre il lavoro. Una possibile limitazione del metodo di lisi dei globuli rossi è la presenza di contaminanti residui derivati ​​da lisati globuli rossi, che potrebbe bloccare la chiara osservazione di trasformazione nelle prime quattro settimane di infezione EBV. Tuttavia, questi detriti sarà progressivamente rimosso con il mezzo modifiche. Un&#39;altra limitazione è la perdita delle crescenti cellule trasformate, ma questo può essere migliorata rimuovendo la metà del surnatante nelle prime quattro settimane di trasformazione. Un&#39;altra possibile modifica per eliminare le impurezze piùfrequenti cambiamenti medi con maggiori volumi o lavaggio delle linee cellulari due volte con 10 ml di PBS 1x e centrifugazione a 168 xg per 5 minuti per rimuovere le impurità. In sintesi, questo protocollo prevede una semplice strategia per la creazione di B-LCL ad alta efficienza immortalizzazione, riducendo il volume del sangue e di tempo che intercorre tra l&#39;inizio e la crioconservazione di risparmio. Le linee cellulari sviluppate nel presente studio forniscono una fonte preziosa e conveniente di biomateriali effettuare vari studi.

Resting B cells could be transformed by the Epstein-Barr virus (EBV) in vitro into actively proliferating B lymphoblastoid cell lines (B-LCLs). B-LCLs are similar to germ cells in differentiation and development, and the somatic mutation rate of B-LCLs was only 0.3%1, with negligible genetic and phenotypic alteration. Therefore, B-LCLs, as surrogates for peripheral blood mononuclear cells (PBMCs), have substantially accelerated the progress of genomics, transcriptomics and proteomics study of EBV associated diseases2,3. Moreover, B-LCLs also have important application in screening monoclonal antibodies4,5. 
Several methods have been developed for the immortalization of B lymphocytes by EBV. The most major common procedures include the isolation of B lymphocytes and the use of B95-8 cell lines to produce infectious EBV. From isolation to transformation, these methods are divided into three strategies. Lymphocytes are separated from fresh blood by density gradient centrifugation and EBV transformation directly6-8, named density gradient separation method in this study. However, other techniques overcome the difficulty of shipping fresh blood and use a procedure for freezing purified lymphocytes and subsequent thawing and EBV infection9-11. Cryopreserved whole blood can also be used for the isolation of B lymphocytes and transformation12,13. In addition to density gradient centrifugation to separate B lymphocytes, some researchers use a magnetic cell sorter to select B lymphocytes12. However, magnetic separation is an expensive, complex and time consuming process. Although these methods are useful to immortalize B lymphocytes with a high success rate, the need to treat a large number of clinical samples and the relatively small volume of blood require a simpler method for the establishment of a permanent cell line.
Another less commonly used method is using whole blood as the source of nucleated cells for EBV infection and the establishment of B-LCLs. These methods simplify the technique by omitting the separation procedure. Only a small amount of whole blood, either fresh14 or frozen15,16, is sufficient to obtain the cell lines. Unfortunately, there is great variability in success from documents15,16 and several of our validation tests. Furthermore, transformed colonies are hardly recognized under a microscope because of the large contaminating impurities. Thus, a more reliable method for transformation is required.
Based on the above considerations, we have developed a novel method to establish B-LCLs without previous purification of the lymphocytes, named the red blood cell lysis method. This method is convenient, time saving and applicable to a large number of samples. As little as 0.5 mL of whole blood was enough to obtain B-LCLs that are easily obtainable from infants and the elderly without significantly harming their health.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:71px;">Techcomp</td>
			<td style="width:119px;">CT6T</td>
			<td style="width:167px;">Centrifugation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automated Cell Counter&#160;</td>
			<td style="width:71px;">Countstar</td>
			<td style="width:119px;">&#160;IC 1000&#160;</td>
			<td>For cell counting&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">&#160;Epoch Microplate Spectrophotometer</td>
			<td style="width:71px;">BioTek Instruments</td>
			<td style="width:119px;">SN263839</td>
			<td>For measuring the absorbance</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">96 well cell culture cluster</td>
			<td style="width:71px;">Corning Incorporated</td>
			<td style="width:119px;">3599</td>
			<td>Polystyrene plates</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">24 well cell culture cluster</td>
			<td style="width:71px;">Corning Incorporated</td>
			<td style="width:119px;">3524</td>
			<td>Polystyrene plates</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">25cm2 cell culture flask</td>
			<td style="width:71px;">Nest</td>
			<td style="width:119px;">707001</td>
			<td>Polystyrene&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">10270</td>
			<td>Component of B-LCLs medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI Medium 1640</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">31800-022</td>
			<td>For B-LCLs medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Glutamine&#160;&#160;</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">0374</td>
			<td>Component of B-LCLs medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 x streptomycin penicillin solution BioRoYeeBRY-2309</td>
			<td style="width:71px;">BioRoYee</td>
			<td style="width:119px;">BRY-2309</td>
			<td>Component of B-LCLs medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ficoll paque plus</td>
			<td style="width:71px;">GE&#160; Healthcare</td>
			<td style="width:119px;">17-1440-03</td>
			<td>For in vitro isolation of lymphocyte</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PHA-M</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">L8902</td>
			<td>For stimulating lymphocyte proliferation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cyclosporin A</td>
			<td style="width:71px;">Cayman&#160; Chemical</td>
			<td style="width:119px;">12088</td>
			<td>For inhibiting the cytotoxicity effect of T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Red cell lysis buffer</td>
			<td style="width:71px;">Tiangen</td>
			<td style="width:119px;">RT122-01</td>
			<td>For lysing red blood cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MTT</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">0793</td>
			<td>For the detection of cell viability</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td>For freezing cells</td>
		</tr>
	</tbody>
</table>

a simple red blood cell lysis method for the establishment of b lymphoblastoid cell lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

Durante il processo di trasformazione, i cambiamenti morfologici di cellule sono visualizzati al microscopio ottico (Figura 2). Piccoli gruppi di cellule erano chiaramente visibile con il metodo di separazione gradiente di densità dopo 7 giorni di infezione, mentre solo uno spesso strato di frammenti cellulari sono visibili dal metodo di lisi dei globuli rossi. Tuttavia, gruppi di cellule linfoblastoidi sono visibili sotto lo spesso strato di frammenti di cellule di 30 giorni dall&#39;infezione. Con più frequente cambiamento medio, diminuisce detriti cellulari ei cluster cellulari sono chiaramente visibili e presentano la stessa immagine stabilita dal metodo di densità di separazione gradiente. Quando le linee cellulari sono state stabilite con successo, il saggio MTT è stato utilizzato per valutare la fattibilità di B-LCL. Secondo la letteratura 17, B-LCL stabilita con metodi differenti sono stati selezionati per rilevare la vitalità cellulare e 1640 supporto è stato utilizzato come controllo in bianco. I dati indicano che la vitalità cellulare di B-LCL stabilito con il metodo di lisi dei globuli rossi è significativamente superiore alla vitalità cellulare dei B-LCL dal metodo di separazione gradiente di densità (Figura 3). <img alt="Figura 1" src="/files/ftp_upload/55191/55191fig1.jpg" /> Figura 1. Diagramma di flusso di procedura. sangue anticoagulante viene trattato con tampone di lisi dei globuli rossi prima dell&#39;infezione EBV seguita dall&#39;aggiunta di CsA. Le cellule B infettate sono coltivate a 37 ° C in presenza del 5% di CO 2 per stabilire e successivamente espandere B-LCL. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55191/55191fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/55191/55191fig2.jpg" /> Fifigura 2. La visualizzazione dei cellulari morfologiche cambiamenti nel processo di trasformazione EBV. La sinistra è B-LCL stabilito con il metodo di lisi dei globuli rossi con 0,5 ml di sangue periferico, e detriti cellulari diminuendo col passare del tempo. La destra mostra linee cellulari stabilizzate dal metodo di separazione gradiente di densità con 2,5 mL di campione di sangue. barra della scala è di 10 micron. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55191/55191fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/55191/55191fig3.jpg" /> Figura 3. Confronto di vitalità cellulare per B-LCL Fondata con diversi metodi. Ogni barra rappresenta media ± SD. I risultati mostrano che vi era una differenza significativa tra i due metodi (p = 0,014). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55191/55191fig3large.jpg" target="_blank">Per favore clicca qui</a>per visualizzare una versione più grande di questa figura.

Watch this Scientific Journal Video about A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines at JoVE.com

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

Un semplice metodo di lisi dei globuli rossi per stabilire B-LCL stato sviluppato con alta efficienza di immortalizzazione, richiede una piccola quantità di sangue e risparmio di tempo tra l&#39;inizio di crioconservazione.

Un cellulare semplice Red Blood Metodo Lysis per l&#39;istituzione di B linfoblastoidi linee cellulari

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.8K Views.  Chinese Academy of Sciences.  Un semplice metodo di lisi dei globuli rossi per stabilire B-LCL stato sviluppato con alta efficienza di immortalizzazione, richiede una piccola quantità di sangue e risparmio di tempo tra l'inizio di crioconservazione. 

Video: A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

L&#39;esaurimento delle popolazioni di cellule specifiche da deplezione del complemento

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

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Immunologia e infezioni

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Purificazione della popolazione di cellule specifiche per fluorescenza separazione delle cellule attivate (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Istituzione di Epstein-Barr Virus Growth-trasformata linee linfoblastoidi

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Gli antigeni Protetto funzionali globuli rossi dalla membrana innesto di Compact Poligliceroli Hyperbranched

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. 
In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.

Ex Vivo Blood Red emolisi test cellulare per la valutazione degli agenti Endosomolytic pH-sensibile per la consegna dei farmaci citosolico Biomacromolecular

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

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 ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Un semplice citometria a flusso metodo per misurare il glucosio assorbimento e glucosio Expression Transporter per monociti sottopopolazioni nel sangue intero

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Destrano migliora l'efficienza di trasduzione lentivirale di cellule NK primarie Murine ed umane

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Un metodo semplice ed efficace per stabilire NK e linee a cellula T da pazienti con infezione attiva cronica del Virus di Epstein - Barr

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Misurazione di deformabilità e l'eterogeneità delle cellule rosse nel sangue di ektacitometria

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...