Name: assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes
Uploaded: 2011-11-01T13:00:00
Description: Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

pMSCV - AID - I - Thy1.1和pKat2载体，从DG Schatz和pVSV -摹，PRSV修订版的礼物，并pMDLg / pRRE载体是一种从BR脐周礼物。

1。准备样品和细胞<ol><li>执行一个中等规模制备的DNA（midiprep）的过度表达或shRNA包含结构和逆转录病毒包装载体pKat2或慢病毒包装载体pVSV - G，pMDLg / pRRE和PRSV - 冯 25 。使用每个构造中的DNA和6微克4微克pKat2（逆转录病毒感染）或每个pVSV - G，pMDLg / pRRE 1.5微克DNA，PRSV - REV包装载体（慢病毒感染）。 </li><li>准备BOSC 23家媒体。转染，使用两个未经修正的贝科的改良Eagle培养基（DMEM），以及完...

<ol>
	<li>Di Noia, J. M., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+antibody+somatic+hypermutation.">Molecular mechanisms of antibody somatic hypermutation.</a> Annu. Rev. Biochem. 76, 1-22 (2007).</li><li>Peled, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+somatic+hypermutation.">The biochemistry of somatic hypermutation.</a> Annu. Rev. Immunol. 26, 481-511 (2008).</li><li>Longerich, S., Basu, U., Alt, F., Storb, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+in+somatic+hypermutation+and+class+switch+recombination.">AID in somatic hypermutation and class switch recombination.</a> Curr. Opin. Immunol. 18, 164-174 (2006).</li><li>Bardwell, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+somatic+hypermutation+and+reduced+class-switch+recombination+in+exonuclease+1-mutant+mice.">Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice.</a> Nat. Immunol. 5, 224-229 (2004).</li><li>Martomo, S. A., Yang, W. W., Gearhart, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Msh6+but+not+Msh3+in+somatic+hypermutation+and+class+switch+recombination.">A role for Msh6 but not Msh3 in somatic hypermutation and class switch recombination.</a> J. Exp. Med. 200, 61-68 (2004).</li><li>Phung, Q. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+hypermutation+at+G+and+C+nucleotides+in+immunoglobulin+variable+genes+from+mice+deficient+in+the+MSH2+mismatch+repair+protein.">Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein.</a> J. Exp. Med. 187, 1745-1751 (1998).</li><li>Rada, C., Ehrenstein, M. R., Neuberger, M. S., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hot+spot+focusing+of+somatic+hypermutation+in+MSH2-deficient+mice+suggests+two+stages+of+mutational+targeting.">Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting.</a> Immunity. 9, 135-141 (1998).</li><li>Rada, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+isotype+switching+is+inhibited+and+somatic+hypermutation+perturbed+in+UNG-deficient+mice.">Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice.</a> Curr. Biol. 12, 1748-1755 (2002).</li><li>Delbos, F., Aoufouchi, S., Faili, A., Weill, J. C., Reynaud, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+eta+is+the+sole+contributor+of+A/T+modifications+during+immunoglobulin+gene+hypermutation+in+the+mouse.">DNA polymerase eta is the sole contributor of A/T modifications during immunoglobulin gene hypermutation in the mouse.</a> J. Exp. Med. 204, 17-23 (2007).</li><li>Masuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+eta+is+a+limiting+factor+for+A:T+mutations+in+Ig+genes+and+contributes+to+antibody+affinity+maturation.">DNA polymerase eta is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation.</a> Eur. J. Immunol. 38, 2796-2805 (2008).</li><li>Lim, D. S., Hasty, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+in+mouse+rad51+results+in+an+early+embryonic+lethal+that+is+suppressed+by+a+mutation+in+p53.">A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53.</a> Mol. Cell. Biol. 16, 7133-7143 (1996).</li><li>Xiao, Y., Weaver, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+targeted+deletion+by+Cre+recombinase+demonstrates+the+requirement+for+the+double-strand+break+repair+Mre11+protein+in+murine+embryonic+stem+cells.">Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.</a> Nucleic. Acids. Res. 25, 2985-2991 (1997).</li><li>Zhu, J., Petersen, S., Tessarollo, L., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+Nijmegen+breakage+syndrome+gene+NBS1+leads+to+early+embryonic+lethality+in+mice.">Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice.</a> Curr. Biol. 11, 105-109 (2001).</li><li>Luo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+mRad50+causes+embryonic+stem+cell+lethality,+abnormal+embryonic+development,+and+sensitivity+to+ionizing+radiation.">Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 7376-7381 (1999).</li><li>Pavri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation-induced+cytidine+deaminase+targets+DNA+at+sites+of+RNA+polymerase+II+stalling+by+interaction+with+Spt5.">Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5.</a> Cell. 143, 122-133 (2010).</li><li>Lee-Theilen, M., Matthews, A. J., Kelly, D., Zheng, S., Chaudhuri, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CtIP+promotes+microhomology-mediated+alternative+end+joining+during+class-switch+recombination.">CtIP promotes microhomology-mediated alternative end joining during class-switch recombination.</a> Nat. Struct. Mol. Biol. 18, 75-79 (2011).</li><li>Basu, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+exosome+targets+the+AID+cytidine+deaminase+to+both+strands+of+transcribed+duplex+DNA+substrates.">The RNA exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates.</a> Cell. 144, 353-363 (2011).</li><li>Yabuki, M., Fujii, M. M., Maizels, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MRE11-RAD50-NBS1+complex+accelerates+somatic+hypermutation+and+gene+conversion+of+immunoglobulin+variable+regions.">The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions.</a> Nat. Immunol. 6, 730-736 (2005).</li><li>Sale, J. E., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TdT-accessible+breaks+are+scattered+over+the+immunoglobulin+V+domain+in+a+constitutively+hypermutating+B+cell+line.">TdT-accessible breaks are scattered over the immunoglobulin V domain in a constitutively hypermutating B cell line.</a> Immunity. 9, 859-869 (1998).</li><li>Cumbers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+iterative+affinity+maturation+of+antibodies+in+vitro+using+hypermutating+B-cell+lines.">Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines.</a> Nat. Biotechnol. 20, 1129-1134 (2002).</li><li>Papavasiliou, F. N., Schatz, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle-regulated+DNA+double-stranded+breaks+in+somatic+hypermutation+of+immunoglobulin+genes.">Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes.</a> Nature. 408, 216-221 (2000).</li><li>Parsa, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+mutates+a+non-immunoglobulin+transgene+independent+of+chromosomal+position.">AID mutates a non-immunoglobulin transgene independent of chromosomal position.</a> Mol. Immunol. 44, 567-575 (2007).</li><li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+instability+of+V+region+hypermutation+in+the+Ramos+Burkitt's+lymphoma+cell+line.">Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line.</a> Int. Immunol. 13, 1175-1184 (2001).</li><li>Ukai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a:T+mutations+is+dependent+on+cellular+environment+but+independent+of+mutation+frequency+and+target+gene+location.">Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.</a> J. Immunol. 181, 7835-7842 (2008).</li><li>Dull, 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+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J. Virol. 72, 8463-8471 (1998).</li><li>Nakamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+class+switching+of+an+IgM++B+lymphoma+clone+CH12F3+to+IgA++cells.">High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells.</a> Int. Immunol. 8, 193-201 (1996).</li><li>Muramatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+expression+of+activation-induced+cytidine+deaminase+(AID),+a+novel+member+of+the+RNA-editing+deaminase+family+in+germinal+center+B+cells.">Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.</a> J. Biol. Chem. 274, 18470-18476 (1999).</li></ol>

如前所述，多元化的抗体细胞株模型已经成为一种流行的出发点，以确定新的蛋白质，抗体多样化的影响在不同的步骤。我们在座的使用在拉莫斯B细胞病毒感染是击倒或过度表达的蛋白质的方法，然后检查SHM影响。 对于这些研究中，我们利用WT拉莫斯和援助您好拉莫斯细胞。野生型细胞可用于研究目标或表达的援助以及援助产生病变的修复，涉及的因素，而?...

推荐试剂-其中大部分来自其他厂商的同类产品，可代替 。 <table border="1" ><tbody><tr><th>试剂名称</th><th >公司</th><th>目录编号</th><th>评论</th></tr><tr><td > 6，以及明确的TC处理的板</td><td >康宁</td><td> 3516 </td><td ></td></tr><tr><td > 10毫升BD露儿- Loksyringes </td><td >屋宇署医疗</td><td > 309604 </td><td ></td></tr><tr><td> 24孔清晰TC治疗板</td><td >康宁</td><td > 3526 </td><td></td></tr><tr><td > TC - 96明确的平底聚苯乙烯处理微孔板</td><td >康宁</td><td > 3596 </td><td ></td></tr><tr><td > 100毫米的TC处理的培养皿</td><td >康宁</td><td > 430167 </td><td ></td></tr><tr><td > Acrodisc针头式过滤器，0.45微米</td><td >颇尔生命科学</td><td > 4604 </td><td ></td></tr><tr><td >洋菜</td><td > Teknova </td><td > A7777 </tD&gt; <td ></td></tr><tr><td >琼脂糖</td><td > GeneMate </td><td> E - 3120 - 500 </td><td ></td></tr><tr><td >氨苄青霉素</td><td>西格玛</td><td > A0166 </td><td> 100 mg / ml的水</td></tr><tr><td >屋宇署FACSCanto II流式细胞仪</td><td> BD公司</td><td ></td><td >或类似的</td></tr><tr><td> BD猎鹰圆底聚苯乙烯管</td><td> BD公司</td><td > 352054 </td><td >为流式细胞仪</td></tr><tr><td > BOSC 23细胞</td><td > ATCC </td><td > CRL - 11270 </td><td ></td></tr><tr><td > 二氧化碳培养箱37 ° C </td><td ></td><td ></td><td ></td></tr><tr><td > DMEM（贝科的修改鹰的介质） </td><td >西格玛</td><td> D6429 </td><td ></td></tr><tr><td > FBS（胎牛血清） </td><td >双子座生物产品</td><td > 100-106 </td><td ></td></tr><tr><td > FITC标记的α-大鼠CD90/mouse CD90.1抗体</td><td > BioLegend </td><td > 202503 </td><td > FITC标记α- Thy1.1 </td></tr><tr><td > FuGENE 6转染试剂</td><td >罗氏公司</td><td > 11814443001 </td><td></td></tr><tr><td > HEPES缓冲溶液</td><td > Invitrogen公司</td><td > 15630-080 </td><td ></td></tr><tr><td > KAPA高保真DNA聚合酶</td><td > KAPA生物系统公司</td><td > KK2101 </td><td ></td></tr><tr><td > LB肉汤（肉汤 - 卢里亚溶原性）粉</td><td > DIFCO </td><td > 240230 </td><td ></td></tr><tr><td >团真相与和解委员会的shRNA细菌甘油</td><td >西格玛</td><td ></td><td> shRNA的载体</td></tr><tr><td > NCS（新生小牛血清） </td><td >双子座生物产品</td><td > 100-504 </td><td ></td></tr><tr><td> PBS（磷酸盐缓冲溶液） </td><td > Invitrogen公司</td><td> 70011 </td><td >在水中稀释至1X </td></tr><tr><td >聚乙烯α-人IgM抗体</td><td > BioLegend </td><td > 314508 </td><td ></td></tr><tr><td > PGS（青霉素，链霉素，谷氨酰胺溶液） </td><td >双子座生物 - P产品展示</td><td > 400-110 </td><td ></td></tr><tr><td>凝聚胺（hexadimethrine溴） </td><td >西格玛</td><td > 107689 </td><td > 10 mg / ml的水</td></tr><tr><td > PureYield质粒Midiprep系统</td><td > Promega公司</td><td > A2495 </td><td></td></tr><tr><td>嘌呤霉素</td><td>西格玛</td><td > P8833 </td><td > 250微克/毫升在水中。 </td></tr><tr><td > QIAquick凝胶提取试剂盒</td><td >凯杰</td><td > 28706 </td><td ></td></tr><tr><td>拉莫斯（RA 1）细胞</td><td > ATCC </td><td > CRL - 1596 </td><td ></td></tr><tr><td > RPMI - 1640培养基</td><td >西格玛</td><td > R8758 </td><td ></td></tr><tr><td >标二</td><td > Invitrogen公司</td><td > 18064-022 </td><td></td></tr><tr><td > SYBR快速定量PCR试剂盒</td><td > KAPA生物系统公司</td><td > KK4601 </td><td ></td></tr><tr><td > Taq DNA聚合酶</td><td > Invitrogen公司</td><td > 18038-042 </td><td ></td></tr><tr><td > TOPO的助教克隆试剂盒</td><td >体外根</td><td > K4520 - 01 </td><td ></td></tr><tr><td > TRIZOL </td><td > Invitrogen公司</td><td > 15596-026 </td><td ></td></tr><tr><td >向导SV基因组DNA纯化系统</td><td > Promega公司</td><td > A2361 </td><td ></td></tr><tr><td > X - Gal的[5 - 溴-4 - 氯- 3 - indoyl -β- D - galatopyranoside] </td><td > Growcells </td><td > C - 5687 </td><td > 40毫克/毫升，在DMSO </td></tr></tbody></table>

assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes

由V（D）J重组产生的低亲和力抗体的B细胞开始他们的生活。然而，检测的病原体后，变量（五）地区的免疫球蛋白（Ig）基因突变约100,000倍以上的基因组的其余通过体细胞超突变（SHM），高亲和力的抗体 1,2 。此外，类开关重组（CSR）产生一种免疫反应，是需要一个特定的病原体不同的效应功能的抗体。 CSR和SHM发起激活诱导的胞苷脱氨酶（AID），deaminates在DNA胞嘧啶残基产生uracils。这些uracils都容易出错的修复途径形式处理，最终导致突变和重组1-3。 我们目前所了解的SHM和CSR的分子细节来自相结合的研究在小鼠原代细胞，细胞系，细胞- F稀土元素的实验。小鼠模型仍显示许多修复因子（如雄，MSH2，MSH6，Exo1，和聚合酶η）4-10的关键作用的基因敲除的黄金标准。然而，并非所有的基因都为淘汰赛研究适合。例如，几双链断裂修复蛋白敲除embryonically致命或损害B细胞发展11-14。此外，有时在SHM或企业社会责任的一种蛋白的具体功能可能掩盖淘汰赛造成全球的缺陷。此外，由于在小鼠实验中，可以长篇大论，改变细胞系中的单个基因的表达已经成为一种越来越受欢迎的第一步，鉴定和表征候选基因15-18。 拉莫斯- Burkitt淋巴瘤细胞系组成进行SHM -曾流行一种细胞系模型来研究SHM 18-24。拉莫斯细胞的一个优势是，他们有一个内置的方便半定量测量的SHM。野生型细胞表达IgM和，因为他们拿起突变，一些淘汰IgM的表达的突变。因此，化验IgM的荧光激活细胞扫描仪（FACS）损失提供了SHM水平的快速读出。 SHM的定量测量，可通过直接的抗体基因测序。 由于拉莫斯细胞难以转染，我们生产的稳定增加或降低一个人的基因的表达与逆转录酶病毒或慢病毒的结构，包含一个过度表达纸盒或一个短发夹RNA（shRNA），分别感染细胞的衍生产品。在这里，我们描述了我们如何感染拉莫斯细胞，然后利用这些细胞来调查SHM特定基因的作用（图1）。

Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

我们描述了如何执行逆转录病毒或慢病毒感染的过度表达或shRNA包含人类拉莫斯的B -细胞系构造和如何衡量在这些细胞中的体细胞超突变。

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Research

JoVE Journal

Immunology and Infection

Murine Model of CD40-activation of B cells

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

纯净小鼠的B细胞的诱导和评估类开关重组

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Detection of True IgE-expressing Mouse B Lineage Cells

检测真正的IgE的表达小鼠B​​系细胞

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

新生儿初乳喂养前后离散催产素分泌脑核细胞应激与炎症的评估

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

小鼠边缘区 b 细胞剪切流诱导迁移的体外分析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...