This work was supported in part by the Lupus Research Institute, NCI grant CA138617, NIDDK grant DK090978, Charlotte Schmidlapp Award (to E.M.J.), and the Albert J. Ryan Fellowship (to J.K.). We are grateful for the support and instrumentation provided by the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children&#8217;s Hospital Medical Center, supported in part by NIH AR-47363, NIH DK78392 and NIH DK90971.

根据该协会的评估和实验动物国际认证与我们的机构动物护理和使用委员会（IACUC）制定准则无特定病原体条件下进行动物实验。 注：表达同类标记纳入小鼠如CD45.1在任供体或受体动物如果可能的话，因为这允许对供体接枝效率和供体的CD4 T细胞群体的特定扩展的监测。如果考虑使用否则转基因小鼠作为供体或收件人，确保应变正确回交至B6的背景，或传送细胞可能会被拒绝，这将在代表性的结果和讨论部分加以处理更详细。 注：以下步骤详细说明卷收获4捐助BM12小鼠，这应该产生足够的细胞注入小鼠12-16。六到十二周龄BM12小鼠产生大约25％的CD4 T细胞大约100-140万美元的淋巴细胞。如果在启动不同数量的小鼠，或不同的小鼠品系，规模量向上或向下相应。 C57BL / 6小鼠一般给类似的产量与接近只有20％的CD4 T细胞。 注：执行所有的步骤在室温，用RT媒体，以避免冷热冲击，这会损害长期的淋巴细胞活力。执行组织收获，并使用无菌技术在组织培养罩组织处理步骤。在这个协议中的所有媒体是IMDM有10％热灭活FBS，除非另外指出，否则，将被简单地称为“完全培养基”。 1.新方法BM12基因分型小鼠注：这里提供一个简单的限制性酶切为基础的协议进行基因分型，为简单和廉价的替代测序，这是目前唯一公布的基因分型方法对这些小鼠。 <ol><li>隔离从小鼠尾巴基因组DNA。请参照详细的协议鼠标尾部剪裁以前的朱庇特的文章，并从尾部产生的cDNA消化13。 </li><li>执行逆转录聚合链式反应（PCR）14以扩增从MHC-II IA b和 IA一个共同的474 bp的DNA片段BM12使用表 1中所列的引物和热循环条件。 </li><li>执行的限制性消化上〜7微升PCR产物利用酶PsuI或其裂酶（BstX2I，BstYI，MflI，或XhoII），其切割野生型IA b，一个 ，但不是IA BM12突变体。遵循消化由生产，这将指定的水的混合物，缓冲液和酶，和5分钟的温育提供协议。至在37℃下15几个小时。 </li><li>加载消化产物并用溴化乙锭14上运行1％聚丙烯酰胺凝胶30-45分钟。在150V-虽然最佳电压和时间设置将根据所用的PCR凝胶装置而有所不同。可视化DNA带用UV照射。代表性的结果示于图1。 </li></ol><img alt="图1" src="/files/ftp_upload/53319/53319fig1.jpg" /> 图1.代表BM12基因分型结果。 为了鉴定小鼠纯合子IA BM12 / BM12，尾DNA中筛选BM12通过PCR /限制性消化的基因分型（步骤1）。纯合野生型（IA B / B）的DNA产生两个频段在227和247基点（可视化为一根粗带位于〜250基点）;纯合子BM12（IA BM12 / BM12）DNA产量为474 bp的一条带;和杂合子（IA BM12 / B）的DNA产生了两个带在〜250和474基点。 <a href="https://www.jove.com/files/ftp_upload/53319/53319fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.收获捐助细胞<ol><li>使用实验动物管理和使用委员会（IACUC）牺牲供体小鼠-approved小学和中学安乐死方法。例如，安乐死的小鼠窒息用CO 2颈椎脱位。 </li><li>使用无菌技术，收获脾和淋巴结（颈浅，下颌，上臂，腋下，肠系膜和腹股沟）到含有10ml完整的媒体在11-13 15ml试管。 注：请参阅详细的淋巴结清扫术协议16以前的报告- 18。这种模式也成功仅使用小鼠脾细胞转移，但是除了淋巴结显著降低供体小鼠的所需数目。 </li><li>产生的单细胞悬液通过糖化使用从注射器柱塞通过70μm的细胞过滤器收集在步骤2.2淋巴组织。为了获得更好的收益，不超载过滤网使用〜6过滤器每4小鼠进行处理，并冲洗过滤器经常同时处理。从4只小鼠结合组织成两个50ml锥形管中，然后离心细胞5分钟，在400×g离心并倾析上清液。 </li><li>悬浮细胞从两个锥形管在50毫升完全培养基，并转移到新的锥形管中。保持这个管在室温。 注：脂肪附着在管的两侧，并且如果管不更换，最终细胞产率会受到影响。 </li></ol> 3.供体细胞计数注：为了保持整个样本的最高活力，红细胞（RBC）裂解不推荐。 <ol><li>混合的单细胞悬液从步骤2.4井，取出1 ml的，并将其传输到一个单独的50ml锥形。抛开其余的，不变的细胞（49毫升）在室温时计数。 </li><li>加入3毫升氯化铵基于红细胞裂解缓冲液，以1ml的供体细胞样品。轻轻混匀1分钟。摇动，然后填充到50毫升完整的媒体，一次离心5分钟。在400 XG，倒出上清液。 </li><li> （用血球例如，用台盼蓝）悬浮红细胞裂解的细胞在10毫升完整的媒体和计数。结果需乘49，这是剩下的被搁置非裂解样品中的细胞的总数量。放弃红细胞裂解的细胞。 </li></ol> 4.供体细胞标记和/或CD4 + T细胞纯化<ol><li>如果需要的话，净化的CD4 T细胞在此阶段，虽然这不是必需的。此外，如果需要的话，标签的细胞用CFSE，如在19或其它的细胞追踪染料，它的一个例子示出在代表性结果部分的图4。 注：对于CD4 T细胞的纯化，负磁选择被推荐的，因为它可以实现高纯度和如在20中所述叶细胞原封不动高生存能力。重要的是，无内毒素的缓冲器被使用;因此，代替牛血清白蛋白，使分离缓冲液的智慧H 2％FBS和EDTA的推荐的浓度。 </li></ol> 5.注射供体BM12细胞<ol><li>后，在400×g下计数供者淋巴细胞在步骤3（和纯化或标记为在步骤4中，如需要的话），离心机细胞5分钟。滗上清，重悬细胞于PBS中每毫升120万美元淋巴细胞（或30万美元纯化每毫升的CD4 T细胞）。转移细胞到无菌的5毫升的圆底管，或其它无菌管，可以轻松地容纳1ml注射器装配有27.5ģ×13毫米的针。 </li><li>在注射之前，预留供体细胞来自步骤5.1的小样品，在4℃下进行流动染色，以确定CD4 T细胞的供体样品中的百分比。作为使用最低抗体面板（表2）在步骤8中描述的染色这些样本。 注意：如果来自不同的小鼠品系细胞作为单独的供体，这是一个重要的考虑因素，并且如果CD4 T细胞的百分比显着变化，PURification可能需要。 </li><li>混合细胞轻轻地，彻底。这可以通过吹打细胞上下用1ml注射器没有连接的针来完成。混合后，绘制细胞在1ml注射器。去除任何气泡后附加针。保持针同时灌注注射器关有助于维持细胞活力。 </li><li>注入每只小鼠250微升（等于30万淋巴细胞，或者750万纯化的CD4 T每只小鼠细胞）腹膜内，如在21中所述。 注：在这里显示的实验，并在我们以前的工作11，每只小鼠的BM12注入捐赠者以30万总淋巴细胞，而不是传统上使用100万总脾细胞。在我们的实验室未公布的数据，没有什么区别，观察血清中抗dsDNA在以下每只小鼠30亿淋巴细胞注射14天。虽然这种显著减少所需的实验小鼠的数目，肾炎机智的发展H细胞的这个数字还没有评估。 </li></ol> 6.确定接枝效率注1：这部分将描述如何确定的供体细胞移植的程度在受体在第3天，以确定哪些可能已经收到了次优的注射任何小鼠（例如，在一个小鼠的移植物的中所见的&lt;10％所有其它小鼠来自相同组）。这些数据还可以帮助确定是否由转基因小鼠品系细胞在稍后的时间点拒绝（详见代表性的结果部分， 图6）。 注2：本节只有当供，受者使用CD45.1 BM12捐助者和CD45.2 C57BL / 6收件人时，从小鼠不同的同类背景，例如，或者当供体细胞进行标记细胞追踪染料。重要的是，第3天注射后，CD4 T细胞已经历最小膨胀（本身Ë代表性结果部分，图4），所以在接枝度观察到的差异是由于变异注射，不膨胀。 <ol><li>麻醉的小鼠用4％的异氟醚或其他IACUC批准的方法。测试后脚反射，以确保鼠标在进行抽血之前被适当麻醉。 </li><li>使用IACUC批准的方法，如后眼窝穿刺如在22收获100-200微升血液。收集血液到含有抗凝血剂，从个人0.5 ml微量离心管如在材料表中引用的等离子体收集管，其含有冻干二钾EDTA中。采血后，用无菌毛巾或纱布，以确保出血停止，然后将鼠标背部在其家笼在那里将继续，直到最后的组织收成（步骤7）施加温和的压力，鼠标眼。 注意：不要让老鼠无人值守，直到老鼠重新获得足够的意识保持腹卧，并不会返回老鼠给别人的公司，直到完全康复。 </li><li>带来血容量到500微升的21℃的PBS中，并转移到锥形底微量离心管中，然后慢慢下垫200微升21℃的高密度细胞分离溶液与200μl的移液管，小心以最小化两相之间的混合（见材料表推荐的解决方案）。离心细胞，在700×g离心20分钟在20-25℃下用离心机制动器设置为低。 </li><li>除去含有淋巴细胞1毫升吸管和转移到含有800微升冷的完整的媒体新的离心管的顶层。轻轻涡旋混合细胞。离心细胞，在700×g离心5分钟，在4℃和倾析上清液。 </li><li>悬浮细胞在200μl流式细胞仪如使用最小抗体面板（表2），以确定吨步骤8中描述的完整的媒体，转移到96孔U型底的板，和用于流污点他的CD4 T细胞的移植物的PBMC作为的百分比的相对丰度。 </li></ol> 7.最终组织收获注：在结果部分所述的实验中，收获注射的供体细胞后14天（或在某些情况下更短的时间），因为它们集中在TFH细胞和浆细胞的最初发展;然而，由于该模式是SLE的一种慢性GVHD模型中，疾病可在更晚的时间点进行监测。最佳的时间表将取决于所构成的每个实验所研究的问题。 <ol><li>在注射BM12供体细胞（步骤5.4）的后一个预定的时间点，使用IACUC批准的安乐死法牺牲小鼠。窒息用CO 2接着放血建议。颈脱位可以用作安乐死的辅助方法，但是这可能会降低血液抽取收率。 </li><li>润湿小鼠的腹部轻轻用含有70％喷雾瓶乙醇。做一个小的，肤浅的切口，手术剪约为1厘米以上的生殖器。退向胸骨腹部的皮肤，注意要保持腹膜筋膜完整。 注意：通常出现0.5-3毫升腹水在第14天，这，虽然还没有被很好地表征，可以测量和分析作为附加参数疾病患病的小鼠。 </li><li>适合5毫升注射器使用18G的针头。针插入有向向上朝向动物的头，并在15°角到筋膜平面的针腹部的右下象限。定位针尖端附近的盲肠，以帮助防止针越来越堵塞肠而抽吸腹水。 </li><li>小心地旋转在其一侧鼠标，然后慢慢吸取腹水入注射器。一旦腹水已被收回，取出注射器并在syri侧面记录分泌物量，根据容积标记NGE。 </li><li>放电腹水到一个5毫升的圆底管并存储在冰上供以后处理。 </li><li>通过从下腔静脉（IVC）基本上如在22拉伸收获血液。用平淡的镊子，轻轻移动肠子鼠标的左侧，揭露下腔静脉。插入一个27.5ģ针装配到1ml注射器入下腔静脉，并慢慢地吸取400-500微升血液。 </li><li>为了最大限度地减少溶血，缓缓注入血液进入0.5 ml离心血清或血浆收集管。对于抗核抗体ELISA法的后血清分析，血不断在冰上。 </li><li>解剖，将获得额外的相关组织（脾和，如果需要的话，淋巴结和肾脏，尤其是当收集在稍后的时间点和肾小球肾炎将得分）。轻轻放置肠子向后朝向右侧的动物，并拉动胰腺，这是脾脏的主要结缔组织除去脾脏。 </li><li>取出所有剩余的胰腺，然后称重脾脏的解剖后立即高精度平衡，总脾肿大是SLE的小鼠模型中常见的参数。将淋巴组织成单独的1.5毫升管填充用1ml完全培养基上冰。固定在10％中性缓冲福尔马林肾脏或卡扣冻结肾脏购买组织学在23。 </li><li> 2小时内收集的3分钟的离心血液。以10,000×g离心（4℃）。除去血清并储存在-80℃下的ANA通过ELISA以后分析。请参考详细ANA ELISA协议24,25以前的报告。在多个：10-20微升等份商店血清，以尽量减少冷冻/解冻循环，并允许更多数量的未来测定的。 </li><li>离心机腹水400×g离心5分钟。取出用1毫升吸管和分装上清分成几个0.5毫升管。冻结上清液在-80℃下购买抗核抗体（抗核抗体）或其它可溶性炎症介质的分析。 </li><li>悬浮细胞FRACT<html>离子在大约1 ml的完整的媒体和传送200μl到96孔U底板用于流染色（如在第8步）。 </li><li>每个捣烂脾通过70微米的细胞过滤到单独的管和冲洗完整的媒体。悬浮的脾细胞用1ml冷的RBC裂解缓冲液1分钟。并通过摇动轻轻混匀。把音量至10毫升冷的完整的媒体，离心5分钟400 XG，倒出上清液。 </li><li>在5毫升悬浮细胞沉淀完整的媒体和计数使用血球。调节音量，使200微升完整的媒体中含有1-3个细胞。开始染色使用扩展板（表2）来定量供体的CD4 T细胞和接收者B细胞分化和扩展流式细胞仪（步骤8）。 注：根据什么激光，光电倍增管（PMT），和过滤器组的组合是可用的，在T和B细胞分析面板分离成多个面板可能是必要的。 </li></ol>ve_title“&gt; 8。流程染色<ol><li>转印1-3百万脾细胞在200μl的完全培养基成单独的5毫升的圆底管，或到96孔U型底的板的单独的孔中。 注意：使用一个96孔板是染色多个样品，但照顾到板样品在每一个其它公为了防止交叉污染的有效方法。一个96孔板可以相应容纳24个样品。 </li><li>离心机板在500×g离心3分钟，然后轻拂上清液从板到合适的（生物危害）容器。 </li><li>悬浮含有可固定存活率染料在制造商推荐的浓度和纯化的抗CD16 /抗CD32抗体混合物在1微克/毫升的细胞沉淀用100微升流动缓冲液（1％FBS的PBS）中。孵育10分钟，在20-25℃下，再加入100微升冷的完整的媒体解渴活力染料。 注：从患病小鼠的脾细胞中通常含有比较高的数字死亡或濒临死亡的细胞;因此，夹杂物生存力染料的建议避免死亡细胞，导致更清洁，更可靠的数据的非特异性抗体标记。类似地，抗CD16 /抗CD32鸡尾酒包含阻断非特异性荧光标记的抗体通过Fc受体结合。 </li><li>离心机板在500×g离心3分钟，然后轻拂上清液从板进入生物危害容器。悬浮细胞在200μl流动缓冲液中。离心机板在500×g离心3分钟，然后轻拂上清液从板进入生物危害容器。 </li><li>悬浮细胞在50μl的流动缓冲器含有抗体混合物（ 表2）。孵育20分钟，在4℃下，再加入150微升流动缓冲液中。重复洗涤步骤8.4。 </li><li>悬浮细胞用在PBS中100μl的2％多聚甲醛。孵育30分钟，在4℃下，再加入100微升流缓冲区。离心机板在500×g离心3分钟，然后轻拂上清液从板进入生物危害容器。 </li><li> Resuspe次在200μl流动缓冲液，转移到流动管插入或标准流管，在黑暗中增加一个额外的100-200微升流动缓冲液，并储存于4℃，直至获取关于流式细胞仪。 </li><li>获得在流式细胞仪上配有相应的激光器和光电倍增管为选定的抗体的面板内染色的数天。除了记录前向散射的宽度和/或高度来转发散射区域，侧向散射区域，并利用荧光参数的区域。为可靠的结果，获得≥1,000供体细胞的每个WT样品中，或总淋巴细胞中，显示供体细胞，在收集的那么多的事件可能是行不通的最小扩展遗传修饰或以其它方式操纵的小鼠的等价数量。 注：流式细胞术分析中详细的代表性结果部（图3 - 6）中所述。 </li></ol>

<ol>
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The authors have nothing to disclose.

的BM12诱导模型是研究SLE的细胞和分子过程相对容易且有效的方式。过继转移的CD4 T细胞直接针对自身抗原的慢性活化导致TFH，气相色谱B细胞和浆细胞的可通过流来测量仪，如这里所描述的积累。采用这种模式可以方便快捷地询问候选基因和新疗法的自身免疫生发中心的过程这类似于那些发生在SLE患者，最终支配自身抗体的病理积累的作用今后的研究。此外，这里所描述的流式细胞仪分析可用于研...

系统性红斑狼疮（SLE）是一种复杂的自身免疫性疾病用抗核抗体（ANA）的生产和肾小球肾炎prototypically特征。许多其他的后遗症，包括皮肤，心肺，肝和病变与疾病在一些个体相关联。在美国的流行率估计相差很大，从150,000-1,500,000 1,2，特别高的发病率在女性和少数族裔3。虽然SLE的病因已经很难辨别，它被认为是来自各种遗传和环境因素，在系统性自身免疫其中高潮的相互作用出现。 许多动物模型已被用于研究导致病情发作和进展的因素。 SLE的经典小鼠模型包括遗传上易感小鼠品系包括NZB x NZW的F1模型及其NZM衍生物中，MLR / lpr狼疮应变，并且BXSB /雅阿应变，和可诱导系统，例如作为pristane和慢性移植物抗宿主病（慢性GVHD）模型4。自身抗体产生的GVHD模型早期的报告中使用的各种小鼠品系或仓鼠株父到F1转移5 - 8;用于研究狼疮样疾病更常见的方法目前包括DBA / 2父→（C57BL / 6×DBA / 2）F 1，和这里描述的BM12传递模型。每个模型都有自己的警告，但他们一般都有一个共同的一套与人类疾病的临床特点相关的功能。在小鼠模型中最经常报告的参数包括脾肿大，淋巴结病，肾炎，ANA生产，并在细胞水平，T滤泡辅助细胞（TFH），生发中心（GC）B细胞和浆细胞的扩张。 H2 - - AB1 BM12 / KhEgJ（BM12）小鼠，应变我的诱导BM12模型由淋巴细胞从IA BM12 B6（C）过继转移实现dentical到C57BL / 6除了上MHC II类3个氨基酸的取代，插入IA b的C57BL / 6（B6）小鼠。捐助者的CD4 T细胞受援国的装甲运兵车Alloactivati​​on导致慢性GVHD的症状非常类似系统性红斑狼疮。具体地，这些包括扩展供体来源的TFH，扩大受者来源的GC B细胞和浆细胞，和生产抗核抗体，包括抗dsDNA，抗单链DNA，抗染色质，以及抗红细胞抗体9。随着时间的推移，受体小鼠发展与在肾小球，间质性的IgG沉积物，和肾脏10的血管区域相关性肾炎。我们最近证明，类似于人类疾病，也有对I型IFN在该模型11中的关键作用。值得注意的是，对于人的SLE的定义标准包括肾炎的SLE兼容的抗dsDNA的存在下发展的抗体12，这两者都是该小鼠模型的显着的特征。 有SE在自发模型BM12模型veral优势。经典模型发展SLE状标志自发依赖任一杂交小鼠品系，近交小鼠品系不上B6背景，或在B6背景大的基因位点，这使交叉敲除或以其它方式转基因小鼠困难且耗时。与BM12诱导模型中，遗传修饰的小鼠可用作任一供体或受体，从而允许更快速鉴定的细胞区室，其中特定基因可能是疾病重要的。此外，在BM12模型疾病发展的速度要快得多，只需要2个星期，直到抗核抗体的外观相比，数月为最自发模型。此外，在对比的是开发疾病在不同时间点的自发模型中，疾病发作和进展中的BM12→B6模型高度同步。这允许适当尺寸的组群，可以B中的代Ë用于介入或治疗策略在疾病发展的任何阶段。 下面是用于通过过继转移BM12淋巴细胞到C57BL / 6小鼠，或遗传变异体上的B6背景发起SLE状自身免疫的详细协议。此外，我们描述了流式细胞染色方案，用于枚举TFH，气相色谱B细胞和浆细胞，细胞类型与人类疾病相关的。重要的是，这些协议也可以被用于表征疾病在SLE最小鼠模型，并确定TFH，气相色谱B细胞和浆细胞中的其他疾病模型。

<table border="0">
	<tbody>
		<tr>
			<td>B6.SJL-Ptprca Pepcb/BoyJ</td>
			<td>The Jackson Laboratory</td>
			<td>001162</td>
			<td>CD45.1+ BoyJ mouse strain</td>
		</tr>
		<tr>
			<td>B6(C)-H2-Ab1bm12/KhEgJ</td>
			<td>The Jackson Laboratory</td>
			<td>001162</td>
			<td>Bm12 mouse strain</td>
		</tr>
		<tr>
			<td>FastDigest PsuI</td>
			<td>Life Technologies</td>
			<td>FD1554</td>
			<td>Restriction digest enzyme for genotyping</td>
		</tr>
		<tr>
			<td>1X RBC Lysis Buffer</td>
			<td>eBioscience</td>
			<td>00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>GE Healthcare</td>
			<td>SH30228.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Separation Tube (PST)</td>
			<td>BD</td>
			<td>365974</td>
			<td>Blood collection tube with Dipotassium EDTA</td>
		</tr>
		<tr>
			<td>Serum Separation Tube (SST)</td>
			<td>BD</td>
			<td>365967</td>
			<td>Blood collection tube with Clot activator / SST Gel</td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>GE Healthcare</td>
			<td>17-1440-02&#160;</td>
			<td>High density cell separation solution</td>
		</tr>
		<tr>
			<td>Lympholyte-M</td>
			<td>Cedarlane</td>
			<td>CL5030</td>
			<td>High density cell separation solution</td>
		</tr>
		<tr>
			<td>GL-7-biotin</td>
			<td>eBioscience</td>
			<td>13-5902-82&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-BUV395</td>
			<td>BD</td>
			<td>564176</td>
			<td></td>
		</tr>
		<tr>
			<td>CD138-BV421</td>
			<td>BioLegend</td>
			<td>142508</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-BV510</td>
			<td>BioLegend</td>
			<td>100559</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR&#946;-BV605</td>
			<td>BD</td>
			<td>562840</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45.1-BV711</td>
			<td>BioLegend</td>
			<td>110739</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45.2-FITC</td>
			<td>BioLegend</td>
			<td>109806</td>
			<td></td>
		</tr>
		<tr>
			<td>PD-1-PE</td>
			<td>BioLegend</td>
			<td>135206</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19-PerCP</td>
			<td>BioLegend</td>
			<td>115532</td>
			<td></td>
		</tr>
		<tr>
			<td>Fas-PE-Cy7</td>
			<td>BD</td>
			<td>557653</td>
			<td></td>
		</tr>
		<tr>
			<td>CXCR5-APC</td>
			<td>BioLegend</td>
			<td>145506</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Viability Dye ef780</td>
			<td>eBioscience</td>
			<td>65-0865-18</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-BV421</td>
			<td>BioLegend</td>
			<td>100443</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2 ml FACS tube inserts, racked</td>
			<td>USA Scientific</td>
			<td>1412-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon&#8482; Round-Bottom Tubes</td>
			<td>BD</td>
			<td>352017</td>
			<td></td>
		</tr>
	</tbody>
</table>

the bm12 inducible model of systemic lupus erythematosus (sle) in c57bl/6 mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

患病小鼠发展脾肿大在短短的14天，表现出的脾脏2-3次健康小鼠的大小在大容量和细胞构成（图2）的条款。 脾依次选通光散射（FSC-A通过SSC-A），消除双峰（FSC-W或-H通过FSC-A），活细胞（活力染料染色的低），以及CD4 +TCRβ+（ 图中图3A）。供体细胞来自基于CD45.1和CD45.2（图3B，左下）受体细胞区别开来。供...

Watch this Scientific Journal Video about The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice at JoVE.com

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

系统性红斑狼疮的BM12诱导模型（SLE）的C57BL / 6小鼠

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

the-bm12-inducible-model-systemic-lupus-erythematosus-sle-c57bl6

Research

JoVE Journal

Medicine

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

17.1K 次观看.  Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine. The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

视频: The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

幸免神经损伤（SNI）模型引起的机械触感小鼠

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

科研

医学

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

胫骨神经切断术 - 一个标准化的模型失神经诱导小鼠骨骼肌

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

局部或全身药物干预后评估的变化小鼠中挥发性全身麻醉药敏感性

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

神经病理性疼痛小鼠坐骨神经掴型号

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

全身及肝脏血流动力学参数的监测小鼠

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.

Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.

在组织变性/再生和癌症的研究利用小鼠诱导端粒酶等位基因

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导："电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status epilepticus (SE) is frequently selected to recapitulate spontaneous recurrent seizures (SRS). Here we present a protocol of SE induction by intraperitoneal (i.p.) injection of pilocarpine and monitoring of chronic recurring seizures in live animals using a wireless telemetry video and electroencephalogram (EEG) system. We demonstrated notable behavioral changes that need attention after pilocarpine injection and their correlation with hippocampal neuronal loss at 7 days and 6 weeks post-pilocarpine. We also describe the experimental procedures of electrode implantation for video and EEG recording, and analysis of the frequency and duration of chronic recurrent seizures. Finally, we discuss the possible reasons why the expected results are not achieved in each case. This provides a basic overview of modeling chronic epilepsy in mice and guidelines for troubleshooting. We believe this protocol can serve as a baseline for suitable models of chronic epilepsy and epileptogenesis.

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

小鼠颞叶癫痫的 Radiotelemetry 模型及脑电图监测

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...