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>
	<li>On day 2, observe the cell morphology under a microscope. Cells are in good condition when they are bright and round. Add 10 mL complete RPMI 1640 medium.</li>
	<li>On day 4, add 10 mL fresh complete RPMI 1640 medium, and then transfer to a T75 culture flask.</li>
	<li>On day 8, add 20 mL complete RPMI 1640 medium.</li>
	<li>Maintain the cells for 8 days without adding fresh medium, i.e., starvation.</li>
	<li>On day 16, observe cells observed under the microscope. Collect the cells in a 15 mL centrifuge tube, 12 mL/tube.</li>
	<li>Cryo-preserve at -80 &#176;C for 1 h, and then rapidly thaw at 37 &#176;C. Repeat 3 times until the virus particles are released from the cells.</li>
	<li>Centrifuge at 240 x g for 10 min at room temperature.</li>
	<li>Filter the supernatant through a 0.22 &#181;m filter, aliquot, 12 mL/tube, and store at -80 &#176;C.</li>
</ol>
2. Treatment of Whole Blood with Red Blood Cell Lysis Buffer
<ol>
	<li>Add 0.5 mL whole blood anticoagulated by EDTA&#903;K2 to a 15 mL tube.</li>
	<li>Put 1 mL red blood cell lysis buffer (filtered by 0.22 &#181;m filter and thawed to room temperature before use) to the whole blood and mix well. Place the mixture at room temperature for 4 min. Then add 5 - 7 mL 1x PBS quickly to the stop reaction.</li>
	<li>Centrifuge at 240 x g for 5 min at room temperature. White blood cells will precipitate at the bottom of the tube.</li>
	<li>Discard the supernatant, and wash the precipitation by adding 7 mL 1640 basic medium. Centrifuge for 5 min at 240 x g at room temperature. Repeat twice.</li>
	<li>Resuspend the cell pellet in 300 &#181;L transformation medium (RPMI 1640 medium, containing 20% fetal bovine serum and 10 &#181;g/mL phytohemagglutinin (PHA)).</li>
</ol>
3. EBV Infection
<ol>
	<li>Add 60 &#181;L of 20 &#181;g/mL cyclosporin A (CsA) to the cell suspension.</li>
	<li>Rapidly thaw an aliquot of EBV at 37 &#176;C. Add 240 &#181;L EBV to the cell suspension.</li>
	<li>Mix well and place the cell suspension to a 96-well plate, 200 &#181;L per well. To avoid evaporation, add 200 &#181;L 1x PBS to every well around of the 96-well plate. Place the plate in a 5% CO2 incubator at 37 &#176;C.</li>
</ol>
4. Expansion and Cryopreservation of B-LCLs
<ol>
	<li>Change half of the medium once per week for the first month after EBV infection. Only a thick layer of cell fragments is visible under microscope at day 7 (Figure 2). After one month of EBV infection, lymphoblastoid cell clusters are visible under a thick layer of cell fragments under the microscope (Figure 2).</li>
	<li>Change half of the medium every 3 days until the cell clusters are clearly visible under the microscope (Figure 2). Typically, this process takes 10 to 20 days.</li>
	<li>After mixing, pipet the cells and transfer to the 24-well plate. Double the volume of culture medium with complete RPMI 1640 while it turns yellow. This process depends on the rate of cell growth.</li>
	<li>Subsequently, transfer the cells to T25 culture flask. Double the volume of culture medium as it turns yellow until the volume of medium expands to 10 - 15 mL.</li>
	<li>Change medium 24 h before freezing. Centrifuge cell suspension for 5 min at 240 x g. Count cells with a cell counter and freeze at a density of 5 - 10 x 106/mL in frozen stock solution containing 90% FBS and 10% dimethylsulfoxide (DMSO). Freeze at a rate of 1 &#176;C per min to -80 &#176;C and then transfer directly into liquid nitrogen.</li>
</ol>
5. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium Bromide (MTT) Assay17
<ol>
	<li>Prepare a B-LCLs single cell suspension with the 1640 medium containing 10% fetal bovine serum. Seed 3 x 104 cells per well onto 96-well plate, 100 &#181;L per well.</li>
	<li>Place the plate in 5% CO2 incubator at 37 &#176;C for 12 - 16 h.</li>
	<li>Add MTT (5 mg/mL) to wells in which cells are growing and keep away from light for 4 h at 37 &#176;C.</li>
	<li>Centrifuge cell plate for 10 min at 168 x g.</li>
	<li>Remove supernatant and add 100 &#181;L DMSO per well. 
	NOTE: Be careful. Do not touch the needle-shaped dye crystals.</li>
	<li>Shake the plate for 10 min at low speed to fully dissolve the crystals.</li>
	<li>Measure absorption at 570 nm by using a microplate spectrophotometer.</li>
</ol>

<ol>
	<li>Mohyuddin, A., 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=Genetic+instability+in+EBV-transformed+lymphoblastoid+cell+lines.">Genetic instability in EBV-transformed lymphoblastoid cell lines.</a> Biochim Biophys Acta. 1670 (1), 81-83 (2004).</li><li>Hu, V. W., Frank, B. C., Heine, S., Lee, N. 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. L., Kunkel, H. G. <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+Epstein-Barr+virus-transformed+B+cell+lines+for+the+generation+of+immunoglobulin-producing+human+B+cell+hybridomas.">Use of Epstein-Barr virus-transformed B cell lines for the generation of immunoglobulin-producing human B cell hybridomas.</a> J Exp Med. 156 (3), 930-935 (1982).</li><li>Traggiai, E., 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+to+make+human+monoclonal+antibodies+from+memory+B+cells:+potent+neutralization+of+SARS+coronavirus.">An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.</a> Nat Med. 10 (8), 871-875 (2004).</li><li>Hussain, T., Kotnis, A., Sarin, R., Mulherkar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+&amp;+characterization+of+lymphoblastoid+cell+lines+from+patients+with+multiple+primary+neoplasms+in+the+upper+aero-digestive+tract+&amp;+healthy+individuals.">Establishment &amp; characterization of lymphoblastoid cell lines from patients with multiple primary neoplasms in the upper aero-digestive tract &amp; healthy individuals.</a> Indian J Med Res. 135 (6), 820-829 (2012).</li><li>Neitzel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Routine+Method+for+the+Establishment+of+Permanent+Growing+Lymphoblastoid+Cell-Lines.">A Routine Method for the Establishment of Permanent Growing Lymphoblastoid Cell-Lines.</a> Hum Genet. 73 (4), 320-326 (1986).</li><li>Oh, H. 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. B., Nielsen, S. 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. C., Beiswanger, C. M., John, E. M., Satariano, E., West, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+transformation+of+cryopreserved+lymphocytes:+A+resource+for+epidemiological+studies.">Successful transformation of cryopreserved lymphocytes: A resource for epidemiological studies.</a> Cancer Epidemiol Biomarkers Prev. 10 (5), 551-554 (2001).</li><li>Tosato, G., Cohen, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Epstein-Barr+Virus+(EBV)-immortalized+B+cell+lines.">Generation of Epstein-Barr Virus (EBV)-immortalized B cell lines.</a> Curr Protoc Immunol. , 22 (2007).</li></ol>

The authors declare that they have no competing financial interests.

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.

<table><tbody><tr><td>Centrifuge</td><td>Techcomp</td><td>CT6T</td><td>Centrifugation</td></tr><tr><td>Automated Cell Counter&amp;nbsp;</td><td>Countstar</td><td>&amp;nbsp;IC 1000&amp;nbsp;</td><td>For cell counting&amp;nbsp;</td></tr><tr><td>Epoch Microplate Spectrophotometer</td><td>BioTek Instruments</td><td>SN263839</td><td>For measuring the absorbance</td></tr><tr><td>96 well cell culture cluster</td><td>Corning Incorporated</td><td>3599</td><td>Polystyrene plates</td></tr><tr><td>24 well cell culture cluster</td><td>Corning Incorporated</td><td>3524</td><td>Polystyrene plates</td></tr><tr><td>25 cm&lt;sup&gt;2 &lt;/sup&gt; cell culture flask</td><td>Nest</td><td>707001</td><td>Polystyrene&amp;nbsp;</td></tr><tr><td>FBS</td><td>Gibco</td><td>10270</td><td>Component of B-LCLs medium</td></tr><tr><td>RPMI Medium 1640</td><td>Gibco</td><td>31800-022</td><td>For B-LCLs medium</td></tr><tr><td>L-Glutamine&amp;nbsp;&amp;nbsp;</td><td>Amresco</td><td>0374</td><td>Component of B-LCLs medium</td></tr><tr><td>100x streptomycin penicillin solution BioRoYeeBRY-2309</td><td>BioRoYee</td><td>BRY-2309</td><td>Component of B-LCLs medium</td></tr><tr><td>Ficoll paque plus</td><td>GE&amp;nbsp; Healthcare</td><td>17-1440-03</td><td>For &lt;em&gt;in vitro&lt;/em&gt; isolation of lymphocyte</td></tr><tr><td>PHA-M</td><td>Sigma</td><td>L8902</td><td>For stimulating lymphocyte proliferation</td></tr><tr><td>Cyclosporin A</td><td>Cayman&amp;nbsp; Chemical</td><td>12088</td><td>For inhibiting the cytotoxicity effect of T cells</td></tr><tr><td>Red cell lysis buffer</td><td>Tiangen</td><td>RT122-01</td><td>For lysing red blood cell</td></tr><tr><td>MTT</td><td>Amresco</td><td>0793</td><td>For the detection of cell viability</td></tr><tr><td>DMSO</td><td>Sigma</td><td>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 ...

a-simple-red-blood-cell-lysis-method-for-establishment-b

Research

JoVE Journal

Immunology and Infection

14.8K Views.  Chinese Academy of Sciences. 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.

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

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

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.

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

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Cancer Research

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

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.

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

In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

Source: Chang, S., et al. <a href="https://app.jove.com/v/53211">In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System.</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to generate plasmacytoid dendritic cells (pDCs) from common lymphoid progenitor cells (CLPs). Upon co-culturing purified CLPs with &#947;-irradiated feeder cells in the presence of Fms-like tyrosine kinase-3 ligand (FL), the CLPs differentiate into non-adherent pDCs, the presence of which is confirmed using flow cytometry.

Source: Chang, S., et al. In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System. J. ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Differentiation Techniques

Isolation and Purification of B Cells from Human Peripheral Blood

Source: Tzeng, S. J. <a href="https://app.jove.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of B cells from human peripheral blood using biotinylated antibodies specific for non-B cells and streptavidin-conjugated magnetic microbeads.

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of ...