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).

이 연구는 유전학과 발달 생물학 연구소의 윤리위원회의 승인을하고, 프로토콜은 인간의 복지를위한 제도적 지침을 따른다. EB 바이러스의 제조 1 <ol><li> 1 일에, 6 mL의 B95-8 세포 배양 해동 완전 RPMI 1640 매체, 10 % 소 태아 혈청, 2 mM L- 글루타민, 항생제 (100 μg의 / ㎖ 스트렙토 마이신, 100 U / ㎖ 페니실린)이 보충 T25 문화 플라스크. 37 ° C에서 5 % CO 2 배양기에서 배양 플라스크를 놓습니다. </li><li> 제 2 일에, 현미경으로 세포의 형태를 관찰한다. 그들은 밝고 라운드 때 세포는 좋은 상태를 유지하고 있습니다. 10 mL의 완전한 RPMI 1640 배지를 추가합니다. </li><li> 하루에 4 일, 10 mL의 신선한 완전한 RPMI 1640 배지를 추가 한 다음 T75 문화 플라스크에 전송할 수 있습니다. </li><li> 하루 8 일, 20 mL의 완전한 RPMI 1640 배지를 추가합니다. </li><li> 즉, 새로운 배지, 기아를 추가하지 않고 팔일의 세포를 유지한다. </li><li> 날 16 일, 세포를 관찰현미경으로 관찰 하였다. 15 mL의 원심 분리 튜브, 12 ㎖ / 튜브에 세포를 수집한다. </li><li> 1 시간 동안 -80 ℃에서 냉동 보존은-하고 빠르게 37 ℃에서 해동. 바이러스 입자가 세포로부터 해제 될 때까지 3 번 반복합니다. </li><li> 실온에서 10 분 동안 240 × g으로 원심 분리기. </li><li> -80 ° C에서 0.22 μm의 필터, 나누어지는 12 ㎖ / 튜브 및 저장소를 통해 뜨는을 필터링합니다. </li></ol> 적혈구 용해 버퍼와 전혈 2. 치료 <ol><li> 15 ㎖의 튜브에 EDTA · K 2 항 응고 0.5 mL의 전혈을 추가합니다. </li><li> 전혈에 (사용 전 0.22 μm의 필터로 여과하고, 실온에서 해동 된) 1 mL의 적혈구 용해 완충액을 넣어 잘 섞는다. 4 분 동안 실온에서 혼합물을 놓는다. 정지 반응에 신속 7 ㎖의 1X PBS - 그럼 5를 추가합니다. </li><li> 실온에서 5 분 동안 240 × g으로 원심 분리기. 백혈구는 튜브의 바닥에 침전된다. </li><li> 상층 액을 버리고, 7 mL의 1640 기본 배지를 추가하여 침전을 씻는다. 실온에서 240 XG에서 5 분 동안 원심 분리기. 두 번 반복합니다. </li><li> (20 % 소 태아 혈청 및 10 μg의 / ㎖의 피토 헤 마글 루티 닌 (PHA)를 함유하는 RPMI 1640 배지) 300 μL 변환 배지에서 세포 펠렛을 재현 탁. </li></ol> 3. EBV 감염 <ol><li> 세포 현탁액에 20 μg의 / ML의 시클로 스포린 A의 60 μL (CSA)를 추가합니다. </li><li> 빠르게 37 ° C에서 EBV의 나누어지는을 녹여. 세포 현탁액 240 μL EBV를 추가합니다. </li><li> 잘 혼합하고 잘 당 96 웰 플레이트에 200 μL를 세포 현탁액을 배치합니다. 증발을 방지 96 웰 플레이트의 주위에 모든 웰에 200 μL 1X PBS를 추가합니다. 37 ° C에서 5 % CO 2 배양기에서 접시를 놓습니다. </li></ol> B-LCLS 4. 확장 및 냉동 보존 <ol><li> EBV 감염 후 첫 달 동안 일주일에 한 번 매체의 절반을 변경합니다. 만 두꺼운 라예세포 조각의 r은 7 일 (그림 2)에서 현미경으로 볼 수 있습니다. EBV 감염 한 달 후에 임파 세포 클러스터는 현미경 (도 2)하에 세포 단편의 두꺼운 층 아래에 표시된다. </li><li> 셀 클러스터는 누가 뭐라고해도 현미경 (그림 2)에서 볼 수 있습니다 때까지 3 일마다 배지의 절반을 변경합니다. 일반적으로이 과정은 10 ~ 20 일이 소요됩니다. </li><li> 혼합 후, 세포를 피펫 팅하고 24 웰 플레이트에 옮긴다. 이 노란색으로 변하면서 완전한 RPMI 1640 배양액의 양을 두 배로. 이 과정은 세포의 성장 속도에 의존한다. </li><li> 이어서, T25 배양 용 플라스크에 세포를 옮긴다. 15ml의 - 매체의 부피가 10로 확장 전까지는 노랑색으로 배양액 부피를 두배. </li><li> 동결하기 전에 매체 24 시간을 변경합니다. 240 × g에서 5 분 동안 원심 분리기 세포 현탁액. FR 10 × 106 / ㎖ - (5)의 농도로 세포 계수기와 함께 동결 세포 개수90 % FBS와 10 % 디메틸 술폭 시드 (DMSO)를 함유하기 Ozen 원액. -80 ° C에 분당 1 ℃의 속도로 고정 후 액체 질소에 직접 전송합니다. </li></ol> 5. 3- (4,5- 디메틸 티아 졸 -2- 일) -2,5- 디 페닐 테트라 졸륨 브로마이드 (MTT) 분석 17 <ol><li> 10 % 소 태아 혈청을 함유하는 배지 1640와 B-LCLS 단일 세포 현탁액을 제조. 잘 위에 96 웰 플레이트 당 종자 3 × 104 세포, 잘 당 100 μL. </li><li> 16 시간 - 12 37 ° C에서 CO 2 배양기 5 %에서 접시를 놓습니다. </li><li> 세포가 성장하는 우물에 MTT (5 ㎎ / ㎖)를 첨가하고, 37 ℃에서 4 시간 동안 빛에 가까이하지 말 것. </li><li> 168 × g으로 10 분 동안 원심 분리기 세포 판. </li><li> 상층 액을 제거하고 잘 당 100 μL DMSO를 추가합니다. 참고 :주의해야합니다. 바늘 모양의 염료 결정을 만지지 마십시오. </li><li> 완전 결정을 용해 저속으로 10 분 동안 플레이트를 흔들어. </li><li> 570 nm의에 의해에서 측정 흡수마이크로 플레이트 분광 광도계. </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.

우리는 적혈구 용균 전혈로부터 인간 B 세포를 불멸화하기위한 새로운 방법의 개발을보고하고, B-LCLS은 MTT 분석법으로 평가 하였다 설정된 그것의 세포 생존. 결과 적혈구의 용해 방법에 의해 확립 된 B-LCLS의 세포 생존율은 밀도 구배 분리 방법보다 훨씬 더 높은 것으로 나타났다. 적혈구 용해 방법의 주요 장점은 간단하고 높은 성공률과 높은 세포 생존과 영구 세포주를 확립하기위한 혈액 (0.5ml?...

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.

변형 과정에서, 세포의 형태 학적 변화는 광학 현미경 (도 2)에 의해 가시화된다. 세포 파편의 두꺼운 층은 적혈구 세포 용해 방법에서 볼 수있는 반면 작은 셀 클러스터는 감염 후 7 일 후, 밀도 구배 분리법 명확하게 볼 수 있었다. 그러나, 임파 세포 클러스터는 30 일 게시물 감염 세포의 단편의 두꺼운 층 아래에 ​​표시된다. 더 자주 변화 매체로, 세포 파편...

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

B-LCLS을 설정하기위한 간단한 적혈구 용해 방법은 소량의 혈액을 필요로하고 동결 보존 개시로부터 시간을 절약 불멸화 높은 효율로 발전되었다.

B 임파 세포 라인의 설립에 대한 간단한 적혈구 용해 방법

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 조회수.  Chinese Academy of Sciences.  B-LCLS을 설정하기위한 간단한 적혈구 용해 방법은 소량의 혈액을 필요로하고 동결 보존 개시로부터 시간을 절약 불멸화 높은 효율로 발전되었다. 

비디오: 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.

보완 고갈에 의해 특정 세포 집단의 고갈

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

연구

면역학 및 감염병학

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.

형광 활성 세포 정렬 (외과)에 의해 특정 세포 인구의 정제

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.

엡스타인 - 바 바이러스 성장 변화 Lymphoblastoid 전지 라인 구축

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.

항원은 컴팩트 Hyperbranched Polyglycerols 중 이식 막으로 기능 적혈구를 보호

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.

Biomacromolecular 일반 의약품의 Cytosolic 전송 산도 - 응답 Endosomolytic 에이전트의 평가에 대한 예 VIVO 레드 블러드 세포 용혈 분석

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.

탯줄 혈액에서 전구체 B-세포 하위 집합의 절연

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.

간단한 유세포 방법은 전체 혈액에서 단핵구 부분 집단에 대한 당 흡수 및 포도당 운송업자 발현을 측정하는

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.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

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.

NK과 만성 활성 엡 스타인-바 바이러스 감염을 가진 환자에서 T 셀 라인을 설정 하는 효율적이 고 간단한 방법

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.

Deformability 및 Ektacytometry에 의해 혈액이 질 레드 셀 측정

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