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），以及完全DMEM。为了使完全DMEM，添加1％HEPES缓冲液，1％青霉素，链霉素，谷氨酰胺（PGS），和10％新生牛血清（NCS）一瓶的DMEM。 </li><li>准备拉莫斯媒体。对于感染的步骤，完成一瓶的RPMI加入的1％HEPES，1％的PGS，10％胎牛血清（FBS），RPMI - 1640培养基（RPMI）。对于单细胞播种步骤，MAKE“空调”的媒体拉莫斯细胞生长在完全RPMI 2天，纺纱400 X克细胞4分钟，过滤取上清液用0.2微米的过滤器进入无菌瓶。其他完整的媒体一样，条件培养基保存于4 ° C直到需要。 </li><li>拆分3 × 10 6 BOSC 23到100毫米的钢板在10毫升完全DMEM细胞，细胞转染前一天，将在50-60％汇合，转天。使每个向量计划转染细胞板。重要的是时间过长，这些细胞没有被保存在文化;使用低通过数量最近解冻的细胞或细胞转染和感染的措施，以确保良好的感染。 </li><li>包括控制你的实验。对于转染和感染，创建一个阴性对照（未转染或未受感染的细胞）和阳性对照（病毒表达GFP和嘌呤霉素抗性基因）。 shRNA的敲下来experiments，感染与杂乱或无关的shRNA控制良好。 </li></ol> 2。 BOSC转染细胞23 <ol><li>未经修正的DMEM和完全DMEM瓶温在37 ° C。 </li><li> BOSC 23细胞中删除的媒体，并与5毫升完全DMEM取代。 37 ° C培养箱，直到返回的细胞在步骤2.6中使用。 </li><li>暖FuGENE 6瓶在室温下5分钟，然后简要涡（&lt;1秒）。 </li><li> 1.5 ml离心管分装成600μL，未经修订的DMEM。 </li><li>未经修订的DMEM 600μL，涡旋简要（&lt;1秒）稀释成30μL，FuGENE 6，然后在室温下孵育五分钟。小心吸管直接进入媒体。不要让FuGENE 6触摸管双方。 </li><li> 6微克构造，无论是4微克pKat2（逆转录病毒感染）或1.5微克每pVSV - G pMDLg / pRRE，PRSV - REV（慢病毒感染）的DMEM / FuGENE 6混合，涡旋BRI<html> EFLY（&lt;1秒），然后在室温下孵育25分钟。 </li><li>添加BOSC 23细胞的DNA / DMEM / FuGENE 6混合和回报为24小时37 ° C培养箱。 </li><li> 24小时后，加入5毫升完全DMEM转染细胞和回报为24小时，37 ° C培养箱。 </li></ol> 3。收获的病毒颗粒<ol><li>收获转BOSC 23细胞用10 mL注射器媒体10毫升。 </li><li>附加0.45μm的过滤器，注射器和过滤到一个干净的15 mL聚丙烯管的媒体。 </li><li>立即使用病毒的媒体为1毫升分装在2 mL低温瓶或冻结在-80 ° C。 </li><li>收获后的病毒媒体，确认，高效的转染阳性对照细胞发生流式细胞仪。这也可以为所有样品，如果结构包含一个荧光（如GFP）或细胞表面标志物（如Thy1）。 </li></ol>书名“&gt; 4。感染B细胞<ol><li>种子2毫升2 × 10 5细胞/毫升完全RPMI拉莫斯细胞到感染前24小时，6孔板。再次，一定要使用低传代次数最近解冻的细胞或细胞，以确保良好的感染。 </li><li>如果使用冷冻的病毒等份，病毒的媒体迅速解冻在37 ° C。 </li><li>混合3μL聚凝胺10毫克/毫升1毫升病毒媒体。对聚凝胺终浓度应总量的3毫升10微克/毫升。 </li><li>细胞和病毒/聚凝胺混合拌匀吹打。如前所述较早，以及应被用来作为一个未受感染的控制;加入1 mL完全RPMI和3μL，聚凝胺这口井，而不是1毫升病毒。请务必感染匹配的阴性对照。我们也传染以及含有GFP的载体，流式细胞仪，以确定感染效率。 </li><li>在室温为90分钟，离心3000 X克在6孔板。 </li><li>返回板块37℃培养箱中培养48小时。 </li><li> 48小时后，400 X克降速感染的细胞在室温下4分钟在15毫升的聚丙烯管。吸取出媒体，确保不扰乱细胞沉淀。在新鲜的RPMI重悬细胞。 </li><li>确认感染流式细胞仪阳性对照样品的效率。像以前一样，可以为您的样品，如果结构包含一个荧光（如GFP）或细胞表面标志物（如Thy1）。 </li><li>开始选择的单元格区域（如适用）。我们的shRNA包含结构中都含有嘌呤霉素抗性卡带。我们选择shRNA的感染拉莫斯细胞，用0.25μg/ mL的嘌呤。我们发现，拉莫斯细胞的衍生工具，有时显示改变到嘌呤霉素的敏感性，因此，我们执行杀以优化每个衍生嘌呤浓度的曲线。也可治疗嘌呤霉素未受感染的控制细胞，以确认选择是有效的的。 </li></ol> &lt;P类=“j​​ove_title”&gt; 5。单细胞种植 单播可以在以下两种方式之一进行：排序或手动流式细胞仪。 <ol><li>流式细胞仪：一个流式细胞仪分选机板适配器使用种子1细胞/孔在96孔板100μL40％RPMI/40％，空调media/20％FBS预填充。此方法提供了很多更多的单细胞克隆，每盘。 </li><li>手动的细胞计数。 RPMI/40％空调media/20％FBS的40％稀释到10个细胞/毫升的细胞等分。使用10毫升稀释的细胞为每个种子板块。不同的克隆将显示在这个阶段变量恢复，因此它是有用的设立在不同浓度（从3到20个细胞/毫升）的几个板块。使用产生较少的副产物，以避免从多个细胞产生的克隆）。使用多道移液器种子96孔板100μL稀释细胞。 </li><li>放置在37℃培养箱中培养2周的板块。 </li><li>检查个别井undeR的显微镜确认细胞的存在一天后播种。单细胞可以比较难找到，由于需要集中在正确的深度。聚焦在96孔板的底部印以及数字应该有所帮助。细胞很容易发现，因为多数井有细胞流式细胞仪分选机种子。 </li><li> 2周后，越来越多的殖民地，可以勉强看到肉眼看从下面板块。他们要容易得多，在显微镜下看到。殖民地往往一起成长的好，通常在平板的菌落生长在不同的井边缘的边缘。马克井具有强大的细胞生长和转移的细胞在1 mL的RPMI的24孔板，并返回到37 ° C培养箱。 </li><li>保持在1 × 10 5至1 × 10 6细胞/ mL的细胞。更改为4分钟，在室温下旋转400 X克的细胞和消除媒体的媒体。更换用1 mL新鲜的RPMI。 </li><li>经过几个ðays的增长，细胞转移到6孔板，在37℃培养箱继续增长。 </li><li> 3周后，分析IgM的损失（如下所述）的克隆。 </li><li>继续增长2个星期的细胞，然后分析，在5周时间点的克隆。 </li></ol> 6。分析：IgM的损失（3周和5周） <ol><li>分装5 × 10 5细胞，从每口井到1.5 ml离心管和旋转400 X克细胞在室温为4分钟。使用未染色控制细胞的控制和击倒的shRNA井，以及一个样本。 </li><li> 1毫升的PBS洗涤细胞，然后再旋转400 X克在室温为4分钟。 </li><li>在50μL的PBS（未染色控制）或PBS悬浮+ 1:20稀释的α-人IgM抗体PE标记。在冰上孵育1小时。 </li><li>在室温下4分钟旋转400 X克细胞。 </li><li>洗的C英语学习者的两倍，1毫升的PBS和自旋为4分钟，每一次都在室温下为400 X克。 </li><li>删除上清100μL的PBS和悬浮。悬浮染细胞转移一个流式细胞仪管。 </li><li>分析细胞，使用流式细胞仪机。 </li><li> IgM的百分比-从与shRNA的拆装下来的细胞的控制细胞进行比较。如前所述，流式细胞仪测定IgM的损失是一个可靠的半定量拉莫斯细胞SHM措施。 </li></ol> 7。分析：确认击倒定量RT - PCR（5周） <ol><li>分装3 × 10 6细胞，从每口井和旋转400 X克在室温为4分钟。 </li><li>准备等分细胞的RNA。我们使用TRIZOL根据制造商的建议。 </li><li>准备从基因的RNA的准备。我们使用的是2微克的RNA和上标II根据制造商的建议。 </li><li>执行定量PCR使用基因confir米敲下来。我们作为一个正常化的控制，并在10μL的反应总体积为SYBR FAST的qPCR试剂盒GAPDH的。 </li></ol> 8。分析：基因组测序（5周） <ol><li>分装5 × 10 6细胞，从每口井和旋转400 X克在室温为4分钟。 </li><li>隔离这些细胞的基因组DNA。我们使用向导SV基因组DNA纯化试剂盒，根据制造商的建议。 </li><li> PCR扩增的Ig V基因19（见表1），使用KAPA高保真DNA聚合酶的利益的任何其他基因的基因组DNA。我们利用聚合酶，因为其低误码率，但任何高保真PCR聚合酶应该是可以接受的的。 </li><li>凝胶纯化PCR产物在1％琼脂糖凝胶使用QIAquick凝胶回收试剂盒。 </li><li>有些聚合酶添加一个单一的脱氧（一）残留物的PCR产物的3&#39;末端，而有的则没有。这3个“A尾巴是必要的TOPO TA克隆的一步。 KAPA高保真聚合酶不留3&#39;一个尾巴。一尾孵化与25μL凝胶纯化PCR产物3μL，10倍的Taq反应缓冲液，1μL10 mM的dNTPs，并在72℃30分钟1单位Taq聚合酶的PCR产物。 </li><li>克隆凝胶纯化的PCR产物使用TOPO TA克隆试剂盒。 TOPO的反应转化到大肠杆菌的产品大肠杆菌 DH5α- T1 ř能干细胞和含有100μg/ mL氨苄青霉素，并根据制造商的建议，补充1.6毫克X - gal的LB /琼脂板的板。 </li><li>发送单个菌落进行测序。 </li><li>执行序列分析，以确定突变的类型和位置。 </li></ol> 9。代表性的成果： 如前所述，我们使用阳性对照病毒载体表达GFP，以及嘌呤霉素抗性基因。我们通常看到的50-75％GFP +细胞，两天转染后。在我们的感染，我们通常看到的50-70％的细胞 +两天后感染，但前选择绿色荧光蛋白。选择完成后，&gt; 95％的细胞GFP +。 为代表的实验中，我们展示过表达的援助或撞倒拉莫斯细胞修复因子的影响。具体来说，我们转了援助逆转录病毒的过度表达载体，通过IRES的网站一起，与逆转录病毒包装载体pKat2 Thy1.1 BOSC 23细胞相连。含有病毒的媒体过滤，然后用来感染拉莫斯细胞。野生型（WT）和援助过表达细胞（援助您好 ）单细胞播种和生长3个星期，在这一点，他们的基因表达分析QRT - PCR（图2）和流式细胞仪IgM的损失（图3 ）。流式细胞仪染色，细胞培养与两个1:20稀释PE标记的α-人IgM抗体以及1:400稀释FITC标记的α-大鼠CD90/mouseCD90.1（Thy1.1）抗体。在另一项实验中中，包含无关的shRNA（对照组）或对高保真的DNA修复，预计防止SHM因素的shRNA慢病毒BOSC 23细胞，然后感染到拉莫斯细胞克隆援助您好。同样，基因表达（图4）和IgM的损失（图5）进行了分析，单细胞克隆后3个星期。由于修复因子被认为是提供对保护在SHM的突变，我们看到在IgM的损失和SHM因素的情况下增加。如果我们下来撞倒在参与生成SHM - 相关突变的一个因素，我们将看到在IgM的损失减少。 正如预期的那样，我们的QRT - PCR结果显示，援助表达援助的过度表达载体（图2）感染细胞后显着增加，而针对性的shRNA对这一因素的存在DNA修复因子下降的水平（图4 ）。因为个人酷龙ES可能会有所不同，你可能会看到一些在基因表达水平的变化。因为基因的表达可以变化，有必要确认您计划进一步分析每个克隆表达。对同一基因的不同的shRNA可能有不同的基因表达的影响一样，所以你应该屏幕几个的shRNA，以确定其中影响最大的。也可能被击倒在蛋白质水平证实，免疫印迹。同样，单个克隆IgM的损失程度可能差别很大。一些克隆可能会表现出“中奖”的效果，IgM的突变发生的最早的细胞分裂之一-例如，你会看到&gt; 50％的IgM损失，仅仅是因为细胞成为IgM抗体-在两细胞阶段。这些克隆的影响最小化的几个单细胞克隆的中位数计算。 <img alt="图1" src="/files/ftp_upload/3573/3573fig1.jpg" /> 跨示意图如图1。染，感染和分析IgM的损失。有关详细信息，请参阅文本。 <img alt="图2" src="/files/ftp_upload/3573/3573fig2.jpg" /> 图2。拉莫斯细胞的援助表达。 WT和援助您好拉莫斯细胞单细胞克隆和生长3个星期，在这点在单个克隆援助表达QRT - PCR测定。 GAPDH的表达被用于正常化。 WT设置为1。中值表示。 *表示p值&lt;0.01，计算为一尾学生t检验。 <img alt="图3" src="/files/ftp_upload/3573/3573fig3.jpg" /> 图3。增加援助您好细胞IgM的损失相比，野生型细胞。在3个星期的时间点的流式细胞仪，表面IgM测定WT 和援助您好细胞IgM的损失的百分比计算。 （一）代表流式细胞仪积了WT克隆和援助您好克隆。的援助过表达Vector已通过IRES的站点链接到Thy1.1的援助，所以Thy1.1表达是用来作为援助表达的替代品。 （二）定量几个WT和援助您好克隆IgM的损失。中值表示。 *表示p值&lt;0.01，计算为一尾学生t检验。 <img alt="图4" src="/files/ftp_upload/3573/3573fig4.jpg" /> 图4。击倒在援助您好细胞修复因子。一个不相干的shRNA控制（控制）或我们的利益的shRNA（shRNA）的，和单细胞种子细胞感染。 3周后，在个别克隆基因表达的QRT - PCR测定。 GAPDH的表达被用于正常化。 WT设置为1。中值表示。 *表示p值&lt;0.01，计算为一尾学生t检验。 <img alt="图5" src="/files/ftp_upload/3573/3573fig5.jpg" /> 图5增加IgM的损失。拆装修复因子水平的援助您好细胞。流式细胞仪在3个星期的时间点，在几个克隆的表面IgM的测定和IgM的损失的百分比计算细胞仍然过度表达的援助。中值表示。 *表示p值&lt;0.01，计算为一尾学生t检验。 <img src="/files/ftp_upload/3573/3573table1.jpg" alt="表1" /> 表1：19的Ig基因引物序列：恒定区Cμ，重链可变区（V H），和轻链可变区（V L） 。

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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>

没有利益冲突的声明。

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 ...

assessing-somatic-hypermutation-ramos-b-cells-after-overexpression-or

Research

JoVE Journal

Immunology and Infection

18.1K 次观看.  Duke University. 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。

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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

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

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 immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

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

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

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

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

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

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

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

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

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

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 and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

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

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

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 antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

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

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 of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

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

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, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

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, ...