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).

该研究得到了中国科学院遗传与发育生物学研究所的伦理委员会，协议遵循人类福利制度的指导方针。 1，EB病毒的制备<ol><li>在第1天，解冻B95-8细胞和培养在6毫升的完全RPMI 1640培养基中，辅以10％胎牛血清，2mM的L-谷氨酰胺，和抗生素（100微克/ mL链霉素，100U / mL青霉素） T25培养瓶中。放置在5％CO 2培养箱中培养瓶在37℃。 </li><li>第2天，观察在显微镜下细胞形态。细胞处于良好状态的时候都亮又圆。加入10 mL完全RPMI 1640培养基。 </li><li>第4天，加入10毫升新鲜完整的RPMI 1640培养基，然后转移到T75培养瓶中。 </li><li>在第8天，加入20毫升完全RPMI 1640培养基中。 </li><li>保持了8天的细胞不加入新鲜培养基， 即 ，饥饿。 </li><li>在第16天，观察细胞在显微镜下观察。收集在15毫升离心管中，12毫升/管细胞。 </li><li>在-80℃下冷冻保存1小时，然后在37℃下迅速解冻。重复3次，直到病毒颗粒从细胞中释放出来。 </li><li>离心在240 xg离心在室温下10分钟。 </li><li>通过0.22微米的过滤器，分装，12毫升/管，并储存在-80℃下进行过滤上清液。 </li></ol> 2.红细胞裂解液全血治疗<ol><li>添加0.5mL的全血用EDTA·K 2抗凝到15毫升管中。 </li><li>把1毫升红细胞裂解液（使用前由0.22微米的过滤器过滤，解冻到室温），以全血拌匀。放置在室温下将混合物4分钟。很快7毫升1X PBS到停止反应 - 再加入5。 </li><li>离心在240 xg离心在室温下5分钟。白血细胞将沉淀在试管的底部。 </li><li>弃去上清液，并通过加入7毫升1640基本培养基洗涤沉淀。在室温下以240 xg离心离心5分钟。重复两次。 </li><li>重悬在300微升变换介质将细胞沉淀（RPMI 1640培养基，含20％胎牛血清和10μg/ mL的植物凝集素（PHA））。 </li></ol> 3. EBV感染<ol><li>添加60微升20微克/毫升环孢菌素A（CSA）的细胞悬浮液。 </li><li>在37℃下迅速解冻EB病毒的等分试样。添加240微升EB病毒的细胞悬浮液。 </li><li>拌匀细胞悬液放置到96孔平板，200微升每孔。为了避免蒸发，添加200μL的1×PBS中，以每个孔周围的96孔板的。放置在一个5％CO 2培养箱板在37℃。 </li></ol> 4，B-LCLS的扩展与冷冻保存<ol><li>每周改变一半培养基一次EBV感染后的第一个月。只有厚厚的拉曳细胞碎片的r是在第7天（ 图2）在显微镜下可见。 EBV感染一个月后，淋巴母细胞簇是下细胞碎片的显微镜（ 图2）下的厚层可见。 </li><li>改变一半培养基每3天直到细胞簇显然显微镜（ 图2）下可见。通常，此过程需要10到20天。 </li><li>混合后，吸管将细胞并转移到24孔板中。双重文化介质的体积完全RPMI 1640，而它变成黄色。这个过程依赖于细胞生长的速率。 </li><li>随后，转移细胞T25培养瓶中。一倍培养基的体积，因为它会变成黄色，直到介质的体积膨胀到10 - 15毫升。 </li><li>冷冻前更改中24小时。离心细胞悬浮液在240×g下5分钟。算用细胞计数器和冷冻细胞以5的密度-在FR 10×10 6 / mL的含90％FBS和10％二甲基亚砜（DMSO）澳升原液。冻结，以每分钟1℃〜-80℃的速率，然后直接转移到液氮中。 </li></ol> 5. 3-（4,5-二甲基吡啶-2-基）-2,5-二苯基四唑鎓溴化物（MTT）测定17 <ol><li>制备B-LCLS单细胞悬浮液用含10％胎牛血清的1640培养基中。每孔到96-孔板种子3×10 4个细胞，每孔100μl。 </li><li>放置在5％CO 2培养箱板在37℃下为12 - 16小时。 </li><li>添加MTT（5毫克/毫升），其中细胞生长的孔，并从光远离4小时，在37℃。 </li><li>离心细胞板在168×g下10分钟。 </li><li>除去上清液，每孔加入100μLDMSO。 注意:要小心。请勿触摸针状晶体染料。 </li><li>振摇10分钟板以低速完全溶解结晶。 </li><li>在由570纳米测量的吸收使用微板分光光度计。 </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.

我们报告的新方法的发展，为通过裂解红血细胞永生化从全血中的人类B细胞，和建立的B-LCLS通过MTT法测定其细胞生存力。结果表明，由红血细胞裂解法建立的B-LCLS的细胞存活率比密度梯度分离方法的高得多。红血细胞裂解方法的主要优点是，它是简单的，并且需要的血液小体积（低至0.5毫升）建立一个永久的细胞系具有高成功率和高细胞生存力。 只有EBV感染之前，还需要一个简单的裂解步骤。未经淋巴细胞先前纯化，细胞损失和细胞损伤被避免。密度梯度分离方法的另一个障碍是溶血。处理血液后在不适当的温度下长期贮存和运输过程中晃动可导致溶血，WHICH将极大地影响淋巴细胞的隔离。其它的研究表明，使用的红血细胞（RBC）裂解导致更高数目的活的PBMC比使用密度梯度分离方法18和淋巴细胞亚型基本上不19影响的。用密度梯度分离法，这是昂贵的和繁琐的相比，红血细胞裂解方法是非常容易，尤其是用于治疗大量的临床样品和新手更实用。 在研究中，我们通过使用红细胞裂解法的永生化24个样品以100％的成功率。与由冻存淋巴细胞报告的成功率和新鲜分离的淋巴细胞的90-94％变为20 2 - 2.5毫升的血液，这成功率要高得多。同时，我们还转化新鲜（0.5mL）中或在不经分离的引用14,15描述冻结（甚至1mL）中的血液，并在efficien变换的CY比报告16（数据未显示）低得多。 本策略不仅提高了效率，永生化显著，而且还降低了血液建立B-LCLS所需的量。对EBV转化现有的技术使用相对大量的外周血约2 - 2.5毫升血液20分离淋巴细胞而有些则需要10毫升或更多10,21。与此相反，以0.5毫升的全血，以获得与此处描述的方法的永生化细胞系需要的。该方法可以被修改以成功永生化从手指采血刺12，这样可以大大促进从小儿患者和老年保护遗传资源。 此外，通过红血细胞裂解法建立的B-LCLS的细胞活力是比上述密度梯度分离法的高。红细胞升ysis方法避免了在密度梯度离心，这将导致对细胞不可修复损伤的过程中机械离心力。这表明，红血细胞裂解方法是更合适的遗传和功能研究，其尽可能快需要细胞大量越好。我们的方法将减少从文化开始到冷冻保存，时间将节约资源，减少劳动力。 红血细胞裂解方法的一个可能限制是污染从红血细胞裂解物衍生的碎屑，这会阻止变换的清楚观察前四周EBV感染的存在。然而，这种碎片会逐渐与介质的变化去掉。另一个限制是生长中的转化的细胞的损失，但是这可以通过在头四个星期变换的除去上清液的一半得到改善。另一种可能的修改，以除去杂质是更具有较大体积或由10毫升1×PBS中洗涤该细胞系两次，然后在168 xg离心离心5分钟以除去杂质频繁介质变化。 总之，该协议提供了用于以高永生化效率建立的B-LCLS，降低血液体积和保存启动和冻存之间的时间的简单策略。在本研究中开发的细胞系生物材料提供了宝贵的和具有成本效益源开展各项研究。

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.

在转化过程中，细胞的形态变化是通过光学显微镜（ 图2）可视化。细胞的小群分别与7天后感染的密度梯度分离法清晰可见，而只有细胞碎片的一厚层是从红血细胞裂解方法可见。然而，类淋巴母细胞的细胞群是细胞碎片的厚层30天后感染下可见。更频繁更换培养基，细胞碎片减少和细胞簇是清楚可见的和呈现由密度梯度分离方法建立的相同的图像。 当细胞系成功建立，MTT测定法来评价B-LCLS的可行性。根据文献17中 ，通过不同的方法建立的B-LCLS被选择来检测细胞活力和1640培养基用作空白对照。数据表明，与红细胞裂解法建立的B-LCLS的细胞生存力大于从密度梯度分离方法（ 图3）的B-LCLS的细胞活力显著更高。 <img alt="图1" src="/files/ftp_upload/55191/55191fig1.jpg" /> 图1.程序的流程图。抗凝血剂的血液EBV感染接着加入环孢素前用红细胞溶解缓冲液处理。在感染的B细胞在37℃下在5％CO 2的存在下，建立和随后展开的B-LCLS生长。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55191/55191fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55191/55191fig2.jpg" /> 网络连接在EB病毒转化过程中的细胞形态学变化的古尔2.可视化。左侧是B-LCLS由红细胞裂解法用0.5ml外周血建立，并且细胞碎片减少随着时间的推移。右侧示出了由密度梯度分离法，用2.5毫升的血液样本建立的细胞系。比例尺为10微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55191/55191fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55191/55191fig3.jpg" /> 图为不同方法建立的B-LCLS细胞活力3.比较。每个条表示平均值±SD。结果表明，有两种方法（p值= 0.014）之间显著差异。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55191/55191fig3large.jpg" target="_blank">请点击此处</a>查看该图的放大版本。

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

建立B-LCLS一个简单的红细胞裂解法与高效率的永生化开发，需要的少量血液，并节省了时间，从开始到冷冻保存。

一个简单的红细胞溶解法对B淋巴母细胞系的建立

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

14.8K 次观看.  Chinese Academy of Sciences. 建立B-LCLS一个简单的红细胞裂解法与高效率的永生化开发，需要的少量血液，并节省了时间，从开始到冷冻保存。 

视频: 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.

补消耗的特定细胞群的枯竭

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

科研

免疫与感染

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.

特定细胞群的净化荧光激活细胞分选（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.

Epstein - Barr病毒生长转化的淋巴细胞系的建立

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.

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

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.

体外红血细胞溶血含量的生物大分子药物的细胞内交货的评价的pH响应Endosomolytic代理

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.

从脐带血分离的前体B细胞亚群

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.

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

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.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

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.

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

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.

Ektacytometry 血中红细胞异质性的测定

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