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).

This study was approved by the Ethics Committee of the Institute of Genetics and Developmental Biology, and the protocol follows the institutional guidelines for human welfare.
1. Preparation of EB Virus
<ol>
	<li>On day 1, thaw B95-8 cells and culture in 6 mL complete RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (100 &#181;g/mL streptomycin, 100 U/mL penicillin) in a T25 culture flask. Place the culture flask in 5% CO2 incubator at 37 &#176;C.</li>
	<l...

<ol>
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We report the development of a novel method for immortalizing human B cells from whole blood by lysing red blood cells, and its cell viability of established B-LCLs was evaluated by the MTT assay. The results showed that the cell viability of B-LCLs established by the red blood cell lysis method is much higher than that of the density gradient separation method. The major advantage of the red blood cell lysis method is that it is simple and requires a small volume (as little as 0.5 mL) of blood for establishing a permane...

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 />
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		<col />
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	<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.

During the process of transformation, the morphological changes of cells are visualized by light microscopy (Figure 2). Small clusters of cells were clearly visible with the density gradient separation method after 7 days of infection, while only a thick layer of cell fragments are visible from the red blood cell lysis method. However, lymphoblastoid cell clusters are visible under the thick layer of cell fragments 30 days post infection. With more frequent medium change,...

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

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

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 ...

The overall goal of this simple method is to establish B lymphoblastoid cell lines, or B-LCLs with high immortalization efficiency using only a small amount of blood and saving time between initiation and cryopreservation. This is a novel method for immortalizing human B cells from whole blood by lysing red blood cells. B-LCLs provides a valuable source of bio-materials to carry out various studies.The main advantage of this method is that only 0.5 milliliters of blood is sufficient to obtain B-LCLs with immortalization efficiency and high cell viability. To establish B-LCLs by Epstein-Barr virus, or EBV, transforming B lymphocytes, the widely used method into separate peripheral blood mononuclear cells or PBMC, by Ficoll density gradient centrifugation. However, when the sample is less than one milliliter of the red blood cell lyse, the addition will fail.We first had the idea for this method when we that to isolate the PBMC from small red blood cells is difficult using a density gradient separation method. To prepare EBV, on day one begin by thawing B95-8 cells and culture them in a T25 culture flask in six milliliters of complete RPMI 1640 Medium supplemented with 10%FBS, two millimolar of L-glutamine, and antibiotics. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide.On day two, under a microscope, observe the cell morphology. Cells are in good condition when they are bright and round. Add 10 milliliters of complete RPMI 1640 Medium to the culture.On day four, add 10 milliliters of fresh medium and transfer the cells to a T75 culture flask. Then, on day eight, add 20 milliliters more of medium and maintain the cells for eight days without adding fresh medium. On day 16, check the cells under the microscope.Then collection 12 milliliters of cells into 15 milliliter centrifuge tubes. Next, cryopreserve the cells at minus 80 degrees Celsius for one hour and rapidly thaw the cultures at 37 degrees Celsius. Repeat the freeze thaw, three more times until the virus particles are released from the cells.Centrifuge the cultures at 240 times g at room temperature for 10 minutes. Then, filter the supernatant through 0.22 micron filter. Aliquot 12 milliliters of the filtrate per tube and store the tubes at minus 80 degrees Celsius.To a 50 milliliter tube, add 0.5 milliliters of whole blood that has been anticoagulated by EDTA K2.Add one milliliter of red blood cell lysis buffer to the whole blood and mix well. Incubate the mixture at room temperature for four minutes. Then quickly add five to seven milliliters of 1x PBS to stop the lysis reaction.After adding red blood cell lysis buffer to blood, be sure to mix well. Invertation ensures that the reaction is terminated in a timely manner. Next, centrifuge the tube at 240 times g at room temperature for five minute.The white blood cells will precipitate at the bottom of the tube. Discard the supernatant and wash the precipitate by adding seven milliliters of 1640 Basic Medium. Then centrifuge the solution before repeating the wash.With 300 microliters of transformation medium, resuspend the cell pellet. Then to carry out EBV infection, add 60 microliters of 20 micrograms per milliliter of Cyclosporin A to the cell suspension. Rapid thaw an aliquot of EBV at 37 degrees Celsius and add 240 microliters of the virus to the cells.Thoroughly mix the suspension and transfer 200 microliters per well to a 96 well plate. Then incubate the plate at 37 degrees Celsius and 5%carbon dioxide. For the first month after EBV infection, change half of the medium in the cell culture once per week.On day seven, only a thick layer of cell fragments is visible under the microscope. After one month of infection, lymphoblastoid cell clusters are visible under a thick layer of cell fragments. From this point forward, change half of the medium every three days until the cell clusters are clearly visible under the microscope.After resuspending the cells, transfer the suspension to the wells of a 24 well plate. Then depending on the rate of cell growth, when the culture medium turns yellow, double the volume of complete RPMI 1640 Medium. When lymphoblastoid cell clusters are visible to the naked eye, transfer the culture to a T25 culture flask.As the culture medium turns yellow, double the volume of the medium until the volume expands to 10 to 15 milliliters. Before freezing, change the medium 24 hours ahead a time. Then to freeze the cells, centrifuge the cell suspension and use a cell counter to count the cells.With the frozen stock solution containing 90%FBS and 10%DMSO, adjust the suspension to a density of five to 10 times 10 to the sixth cells per milliliter. Then freeze the suspension at a rate of one degree Celsius per minute to minus 80 degrees Celsius before transferring the frozen cells directly into liquid nitrogen. During the process of transformation, the morphological changes of cells are visible by light microscopy.After seven days of infection, small clusters of cells are clearly visible using the density gradient separation method, while only a thick layer of cell fragments are visible from the red blood cell lysis method. 30 days post infection, lymphoblastoid cell clusters are visible under the thick layer of cell fragments. With more frequent medium changes, the cell debris is reduced and the cell clusters are clearly visible and look similar to the culture established by density gradient separation.Finally the MTT assay was used to evaluate the viability of B-LCLs. The results shown here indicate that the B-LCLs cell cultured using the red blood lysis method were significantly more viable than the culture established using the density gradient separation method. Once mastered, the EBV viral transformation process can be done in 30 minutes when it is performed properly.After watching this video you should have a good understanding of how to establish B-LCLs with a simple and time saving red blood cell lysis method without previous purification of lymphocytes. After its development, this technique accelerated the progress of the genomics, transcriptomics, and proteomics study of EBV associated diseases and this application in the production of mononuclear antibodies. Don't forget that working with human pathogens, such as EBV can be extremely hazardous and the precautions should always be taken while performing this procedure.

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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 ...

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 ...