Name: a simple red blood cell lysis method for the establishment of b lymphoblastoid cell lines
Uploaded: 2017-01-14T15:00:00
Description: Watch this Scientific Journal Video about A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines at JoVE.com

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).

Este estudo foi aprovado pelo Comitê do Instituto de Genética e Biologia do Desenvolvimento de Ética e o protocolo segue as diretrizes institucionais para o bem-estar humano. 1. Preparação de vírus EB <ol><li> No dia 1, descongelar células B95-8 e cultura em 6 ml de RPMI completo 1640, suplementado com 10% de soro fetal de bovino, 2 mM de L-glutamina, e antibióticos (100 ug / ml de estreptomicina, 100 U / mL de penicilina) numa balão de cultura T25. Colocar o frasco de cultura em 5% de CO2 ...

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Relata-se o desenvolvimento de um novo método para imortalizar células B humanas a partir de sangue total por lise de células vermelhas do sangue, e a sua viabilidade celular estabelecida de B-LCLS foi avaliada pelo ensaio de MTT. Os resultados mostraram que a viabilidade da célula de B-LCLS estabelecidos pelo método da lise de células vermelhas do sangue é muito maior do que a do método de separação por gradiente de densidade. A principal vantagem do método de lise de glóbulos vermelhos é que é simples e ...

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.

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		<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 o processo de transformação, as modificações morfológicas das células são visualizados por microscopia de luz (Figura 2). Pequenos grupos de células foram claramente visíveis com o método de separação por gradiente de densidade depois de 7 dias de infecção, ao passo que apenas uma camada de espessura de fragmentos celulares são visíveis a partir do método de lise de glóbulos vermelhos. No entanto, grupos de células linfoblastóides são visíve...

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

Um método simples de lise de glóbulos vermelhos para o estabelecimento de B-LCLS foi desenvolvido com alta eficiência de imortalização, requerendo uma pequena quantidade de sangue e de economia de tempo desde a iniciação à criopreservação.

Um glóbulo vermelho simples método de lise para o estabelecimento de linhas celulares linfoblastóides B

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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