The authors thank Dr. Michael N. Sack (National Heart, Lung, and Blood Institute) for support and discussion, Ms. Ann Kim for optimizing the B cell isolation protocol, Dr. Joseph Brzostowski for his help with microscopy, and Ms. Mirna Pe&#241;a for maintaining the animals used. This study was supported by the Intramural Research Programs of the National Institutes of Health, National Institute of Allergy and Infectious Diseases, and National Heart, Lung, and Blood Institute.

所有动物实验按照LIG-4的动物的协议，这是经NIAID动物护理和使用委员会进行。 1.试剂的制备<ol><li>制备磁分离（MS）的缓冲液:补充有0.5％BSA和2mM EDTA或补充有0.5％BSA的自动分离溶液的PBS（pH7.2）中。无菌过滤器（0.22微米），并储存在2-8°C。 </li><li>制备RF10培养基:RPMI1640培养基补充有10％热灭活胎牛血清，50U / ml青霉素，50μM链霉素，1mM的丙酮酸钠，2mM的L-谷氨酰胺，0.1mM非必需氨基酸，50μM2-巯基乙醇，10mM的HEPES。用消毒试剂。无菌过滤器并储存在2-8°C。 </li><li>制备线粒体压力测试介质:补充有1mM丙酮酸钠，2mM的L-谷氨酰胺和25mM葡萄糖XF基础培养基。 </li><li>准备糖酵解压力测试介质:XF基本培养基supplemen泰德了2mM L-谷氨酰胺。 </li><li>同时调整线粒体和糖酵解压力测试介质的pH到7.4，无菌过滤器和储存在2-8℃。温暖的媒体至37°C和重新调整pH之前使用（进行测量pH值在37°C）。 注意:使用XF介质，它缺乏碳酸氢盐，是至关重要的。由于在细胞外磁通分析器气氛仅包含环境CO 2，在介质中的任何碳酸氢盐，结合后释放糖酵解的副产品质子，将解离，以形成二氧化碳和水。 的 CO 2将在实验过程中脱气，引起pH值的漂移，将模糊糖酵解酸化信号。 </li><li>寡准备:准备10毫米DMSO原液;保存在-20℃。 </li><li>准备2,4-DNP:准备在DMSO 1M的原液;保存在-20℃。 </li><li>准备抗霉素A:准备10毫米DMSO原液;保存在-20℃。 </li><li>鱼藤酮准备:准备10毫米小号在DMSO滴答的解决方案;保存在-20℃。 </li><li>准备葡萄糖:溶在糖酵解压力测试介质葡萄糖以形成250mM的溶液;准备每次实验前新鲜和不冻结。 </li><li>制备2-DG:将固体2-DG在糖酵解压力测试介质中以形成一个500毫溶液;准备每次实验前新鲜和不冻结。 </li></ol> 2.收获脾和淋巴结小鼠<ol><li>安乐死用CO 2窒息之后颈脱位鼠标。 </li><li>喷用70％乙醇鼠标。 </li><li>对于随后的T细胞的分离，收获了脾，颈浅的，肤浅/渊腋，下颌骨，腹股沟，和肠系膜淋巴结。 </li><li>对于后续的B细胞分离，只收获了脾脏。 注:使用生物安全柜，并保持收获的器官在PBS或RF10媒体上的冰上，直到处理。使用无菌实验室器皿及无菌技术为所有的处理和ISOL通货膨胀步骤。置于冰上所有缓冲区和媒体，以增加隔离效率，减少细胞死亡。 </li></ol> 3.组织处理<ol><li>放置一个70微米的细胞滤网超过50ml锥形管和器官转移到过滤器。对于T细胞的分离，结合所有的器官和B细胞隔离变换仅脾脏。 </li><li>从无菌3毫升注射器使用柱塞，通过过滤醪组织。糖化过程中，确保过滤器和器官是在任何时候都湿润，并根据需要加入MS缓冲弄湿。这将既保持细胞生存力高和防止过滤器的堵塞。 </li><li>捣碎后，取5〜10毫升缓冲MS的过滤器，以增加细胞恢复。离心该悬浮液在400×g离心在4℃下5分钟。 （进行所有进一步离心这些条件下，除非另有说明）。 </li><li>倒出液体并重新悬浮于5毫升的ACK红细胞裂解缓冲液沉淀。 INCU软化在冰上5分钟，以裂解红血细胞（红血球需要被去除，因为它们与磁分离干扰）。添加10-20毫升MS缓冲和离心。 </li><li>放置一个30微米的细胞过滤器在15毫升的锥形管和总理就用1毫升MS缓冲（这将过滤从红细胞裂解清除杂物）。倒出从离心管中的液体，重悬沉淀在5ml MS缓冲，并使之通过过滤器。洗净初始管用4ml MS缓冲增加细胞恢复，并用它来冲洗过滤器。 </li><li>使用血细胞计数器或自动细胞计数器，确定细胞的总数。此细胞数将被用于确定如果脾细胞是在随后的胞外通量测定法中使用在第4部分所需磁选试剂的量，预留的细胞的适当数量的在这个时候。 </li><li>离心悬挂和进行磁选。悬浮脾不使用对于在5毫升RF10 B细胞的分离和保持在冰上，直到外通量分析。 </li></ol> 4，B细胞和幼稚的CD4 + T细胞磁选<ol><li>确定生物素抗体混合物，抗生物素微珠，和MS缓冲器的必要容量。使用10 8个细胞以下几卷作为指导和放大或缩小是必要的。 注:孵育样品在冰箱中，而不是在冰上自孵育在冰上可减少抗体结合效率和可能需要较长的温育时间。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> 试剂幼稚CD4 + T细胞分离 </td><td> 为10 8个细胞体积 </td></tr><tr><td> （适当的比例） </td></tr><tr><td>生物素标记抗体鸡尾酒</td><td> 100微升</td></tr><tr><td> MS缓冲区第一孵化</td><td> 400微升</td></tr><tr><td>抗生物素微珠</td><td> 200微升</td></tr><tr><td> CD44微珠</td><td> 100微升</td></tr><tr><td> MS缓冲区第二培育</td><td> 200微升</td></tr><tr><td colspan="2">孵育在4℃下的生物素 - 抗体混合物和细胞5分钟。添加抗生物素微珠，CD44微珠，质谱缓冲，并在4℃下孵育15分钟。 </td></tr></tbody></table> 表1:T细胞分离的卷和指令。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> 试剂B细胞分离 </td><td> 为10 8个细胞体积 </td></tr><tr><td> （适当的比例） </td></tr><tr><td>生物素标记抗体鸡尾酒</TD&gt; <td> 100微升</td></tr><tr><td> MS缓冲区第一孵化</td><td> 400微升</td></tr><tr><td>抗生物素微珠</td><td> 200微升</td></tr><tr><td> MS缓冲区第二培育</td><td> 300微升</td></tr><tr><td colspan="2">孵育在4℃下的生物素 - 抗体混合物和细胞15分钟。添加生物素抗体混合物和MS缓冲器的适当体积，并在4℃下孵育该混合物15分钟。添加抗生物素微珠的适当体积和MS缓冲液在4℃下孵育该混合物15分钟。第二次温育之后，填充与MS缓冲器和离心试管。 </td></tr></tbody></table> 表2:B细胞隔离卷和指令。 <ol start="2"><li>在重悬缓冲MS颗粒:500微升手动分离，3-4毫升自动分离。 </li><li>无论是用手动或自动离职继续如下: <ol><li>手动分离: <ol style="margin-left: 40px;"><li>插入一个LS柱进入分离磁铁，放置在下面一个无菌的15毫升锥形管通过用3ml的MS缓冲液洗涤，收集通过素柱的流动，和。丢弃通过流。 </li><li>细胞悬液转移至涂底漆的LS柱，并允许它通过;洗用3ml的MS缓冲和传递此通过该柱，以及温育过程中使用的管。洗脱液将包含纯化的细胞。 B细胞分离，在第二时间经过纯化的细胞通过该柱到一个新的15ml试管中，并重复洗涤步骤。这将增加的纯度和产率。 </li></ol></li><li>自动分离: <ol><li>开关上的自动分离器，并检查运行缓冲液，冲洗液和70％乙醇的含量。确保废物是在开始分离之前空。 </li><li>根据T的管的尺寸选择合适的冷藏架他未纯化样品（ 例如 ，15毫升）。为了保持整个分离过程可行的细胞，使用已在2-8℃下预先冷却3-4小时机架，或直到冷却剂变成固体。 </li><li>安装在自动分离装置的样品台的冷冻架上。 </li><li>放置在插槽A1，对于在时隙B1中阴性部分的管样品管，以及用于在C1上的正部分的管。如果收集多个样品，放置在下一列（A2，B2，C2， 等等 ）的其他管中。 </li><li>选择在触摸屏上的分离标签，并指示在机架管的布置。从分离菜单中，选择"耗尽"程序，并完成与适当类型洗涤程序周期（参见下面的注释）。 注意:"耗尽"计划执行使用机器的最敏感的模式，优先纯度枯竭。此外，有三种不同的洗涤选项:QUICķ漂洗，和睡眠。如果样本是从同一源和将被组合，选择快速冲洗。如果样本将被分开，选择冲洗。选择睡眠选项，如果没有进一步的隔离将开展那一天，如果机器将被关闭以下隔离</li><li>按"运行"，启动隔离和提示时确认缓冲水平。程序结束后，负分数将包含纯化的细胞。 </li></ol></li><li>填充锥形管高达15毫升MS缓冲和离心机。 </li><li>倒出该MS缓冲液和悬浮细胞在5ml RF10介质。保持细胞在冰上，直到外通量分析。 注:在理想情况下，胞外通量测定法，应立即在分离后进行。然而，在初步实验中隔离与新鲜分离的人的3小时内使用的细胞中观察到在测定结果没有显著差异。 </li></ol></li></ol>注意:此处所概述的协议是用于仪器的96孔格式。卷将需要进行调整，如果使用另一种格式。 <ol><li>传感器盒的水化<ol><li>抬起传感器盒，填充用200μl的校准溶液的效用板的各孔，并降低传感器盒上的效用板，浸入传感器中的校准溶液。 注:验证校准解决方案级别足够高，以保持淹没传感器。墨盒港口关联O 2和H +每口井的荧光;这些都需要以水合为他们正常工作。 </li><li>放置盒式磁带在37℃培养箱中未补充的 CO 2或氧/氮。执行所有进一步孵化这些条件下，除非另有说明。孵育墨盒一夜之间水合物。 </li></ol></li><li> Prepar胶涂层板的通货膨胀<ol><li>在0.1M碳酸氢钠制备2.5 22.4微克/毫升粘合剂溶液中，pH为8.0（碳酸氢盐为粘合剂吸附最适pH）。 </li><li>应用25微升溶液到测定板的每个孔中，并在室温下在工作台上孵育20分钟。 </li><li>抽吸粘合剂和洗涤各孔两次使用200微升无菌水。等到井接种细胞前，干燥。 </li></ol></li><li>在胶粘剂涂层板的种子细胞<ol><li>离心细胞以在200×g离心5分钟，室温。在5毫升的合适的测定介质的悬浮细胞（见上文线粒体应力和葡萄糖应力介质的制剂）。再次离心细胞，并重新悬浮在测定培养基中的所需浓度（悬浮体积将取决于浓度;每个孔将包含180微升细胞悬浮液）。 注意:只要适当测定培养基和化合物s的所使用的，细胞外的磁通分析仪可以同时运行在同一板上的线粒体和糖酵解压力测试。 </li><li>板180微升细胞悬浮液在每个孔中。使用井A1，A12，H1和H12为背景温度校正:加180微升测定培养基中这些孔（无细胞）。 </li><li>在37℃下孵育板25分钟。 </li><li>离心板在200×g离心5分钟，没有制动，以确保所有细胞已完全附着。目视确认该细胞被稳定地附着于培养物表面，形成单分子层，通过在显微镜下观察。转移板回培养箱另外的30分钟。 </li></ol></li><li>化合物的浓度10倍的制备和喷油港口装载<ol><li>对于线粒体压力测试，准备2.5毫升10μM寡，1毫米DNP，和10μM鱼藤酮和10μM抗霉素A，所有的线粒体压力试验mediu的混合物米 注:2,4-DNP的浓度是基于先前发表的研究; 21，这是一般的准则，但解偶联剂的用于使用应该由研究人员来确定每种细胞类型的浓度。 </li><li>对于糖酵解压力测试，准备2.5毫升250毫米的葡萄糖，10μM寡，而在糖酵解压力测定介质500毫摩尔2-脱氧葡萄糖（2-DG）的。 </li><li>温暖10倍溶液至37℃，并调节pH至7.4。 </li><li>加载化合物成使用多通道移液器和装载导轨盒的合适注射器端口，具体如下: <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 港口 </td><td> 线粒体应力分析 </td><td> 糖酵解应力分析 </td></tr><tr><td>一个</td><td> 20微升寡</td><td> 20微升葡萄糖</td></tr><tr><td>乙</td><td> 22微升2,4-DNP </td><td> 22微升寡</td></tr><tr><td> C </td><td> 25微升抗霉素A /鱼藤酮</td><td> 25μl的2-DG </td></tr></tbody></table> 表3:化合物卷。 注:每个系列的端口（ 例如 ，所有的端口A）必须包含相同的音量。至关重要的是，在一个给定系列中的所有孔中被加载，即使是那些在实验中不使用，否则这些化合物将不会被注入（在未使用的孔中的端口可以装入相同体积的测定培养基的）。 </li><li>孵育盒在设置程序。 </li></ol></li><li>设置外通量分析协议<ol><li>设置了下面的程序（ 见表4）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 步 </td><td> 循环 </td></tR&gt; <tr><td>校准</td><td> - </td></tr><tr><td>平衡</td><td> - </td></tr><tr><td>基准读数</td><td> 3次:混合3分钟，等待0分钟，测量3分钟</td></tr><tr><td>结束循环</td><td> - </td></tr><tr><td>注射端口A </td><td> - </td></tr><tr><td>测量</td><td> 3次:混合3分钟，等待0分钟，测量3分钟</td></tr><tr><td>结束循环</td><td> - </td></tr><tr><td>注射端口B </td><td> - </td></tr><tr><td>测量</td><td> 3次:混合3分钟，等待0分钟，测量3分钟</td></tr><tr><td>结束循环</td><td> - </td></tr><tr><td>注射端口C </td><td> - </td></tr><tr><td>测量</td><td> 3次:混合3分钟，等待0分钟，测量电E 3分钟</td></tr><tr><td>结束循环</td><td> - </td></tr><tr><td>程序结束</td><td> - </td></tr></tbody></table> 表4:项目布局。 注意:在某些情况下， 例如 ，使用活化细胞时，酸化可能持续超过幼稚细胞更长。因为这个原因，它可能有必要在上述程序扩展的"等待"时间或重复在每个测量周期更多次。 </li><li>开始的程序。校准步骤后，（提示时）更换校准板分析板。 注意:使用的软件，它可以指示具有类似条件井组。此外，它是指示哪些井对照孔关键（在这个协议中，它们是在板的四角），并指示哪个孔是空的。有关更详细的，一步一步，协议设置机器和它的软件，制造商的网站，应咨询。 </li></ol></li><li>测量蛋白质含量<ol><li>除去从各孔中剩余的测定培养基而不干扰细胞。 注意:如果它是不方便的蛋白质浓度测定来进行，也可以在-20℃直至分析冻结整个板。如果冻结，继续之前解冻。 </li><li>制备蛋白酶抑制剂的在RIPA裂解介质1倍溶液（100×股票）（足够用于50μl/孔）。 </li><li>添加补充有蛋白酶抑制剂，以每孔50微升的RIPA裂解介质。搅动上5分钟摇动板，然后孵育板在冰上进行30分钟至完全裂解细胞。 </li><li>旋板在200×g离心5分钟，在室温下使脂质和其它分子到板的底部，以便它们不与二辛可宁酸（BCA）测定干扰。 </li><li>根据制造商通过BCA测定测量蛋白质浓度和＃39;的建议。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests. Open Access fees for this article were provided by Agilent Technologies.

的协议中，我们开发了允许纯的和可行的淋巴细胞亚群，其随后在细胞外通量分析中使用不同浓度来评估在糖酵解和线粒体呼吸性能的差异的有效隔离。这个协议是对淋巴细胞专门设计和解决与这些细胞类型，如低基础代谢活性，脆性，低频率的特殊考虑，并且它们不能坚持测定板。因此，与先前公布的协议11,12，我们的方法提供了在免疫学领域工作的研究人员更方便和更好优化的指南。有此协议中的几个关键步骤，包括获得纯净和可行的细胞群体，在最佳汇合电镀细胞，规范外通量分析测量每个蛋白浓度良好。 第i镀既能通过光学显微镜可视化并且还细胞nitial数使用蛋白质浓度测量来量化。细胞数和蛋白质浓度线性相关证实该蛋白浓度确实可以用作一种方法来标准化在不同孔之间的细胞数的变化的影响。 通过标准化ECAR和OCR结果向蛋白质浓度，我们发现，尽管降低细胞汇合时，少至2.5×10 5细胞/孔，可以在最测定中使用而不损害数据质量。然而，可以细胞类型和测定法之间使用不同的细胞的下限。例如，虽然少至1.25×10 5细胞可用于B细胞的OCR测量，甚至2.5×10 5细胞/孔不足以评估相同的细胞类型的糖酵解性能。因此，只要细胞数不是一个限制因素，在以上电镀90％汇合，这大约对应于5×10 5淋巴细胞/孔，是优选的。进一步的优化可能需要时的细胞不同的尺寸，如先前激活和部分分化的淋巴细胞-被使用。另外，使用预先培养的原代淋巴细胞时，有可能是在不同的处理条件之间细胞生存力的变化，这将降低蛋白质浓度的可靠性，因为死细胞数的度量或死的细胞也可以向所测量的蛋白水平。在这种情况下，这可能是有益的流式细胞仪进行细胞外通量测定法之前，通过流活细胞进行排序。 在使用新鲜分离和高度可行的淋巴细胞群我们的分析，我们获得了两个OCR和ECAR测量功能可靠的数据。虽然所有类型的细胞线粒体压力测试类似表现，细胞类型之间存在着明显差异在糖酵解应力测试中观察。例如，相比于脾细胞或B细胞幼稚T细胞的糖酵解性能是低的，并没有在添加寡的变化。这个观察是在与先前发表的研究7,30线，证实了我们的协议的有效性。 总之，我们的方法提供了使用的一个细胞外磁通分析器测试淋巴细胞的代谢活性的有效和方便的方式，它可以在很宽的范围内的免疫学研究的探索活化时，细胞分化，或由于在免疫细胞中的代谢变化是有用疾病的表型如感染，自身免疫和血液恶性肿瘤。

针对抗原的免疫应答是免疫激活和免疫抑制之间的紧密调节平衡。免疫激活驱动响应于所述刺激的快速细胞增殖和迁移，以及细胞因子的产生，抗体分泌和增加的吞噬作用，而免疫抑制简单地抑制这些事件，因此在防止不必要的免疫应答1-6重要。最近的研究已经表明，免疫细胞的活化状态和各种代谢途径7的活性之间存在直接的联系。免疫细胞可以通过开，关能源生产途径休息和激活状态之间切换。此外，已经观察到不同的免疫细胞类型可使用不同的代谢策略激活期间的燃料其增加的能源需求。例如，尽管T淋巴细胞的活化引导细胞进入一个几乎完全糖酵解状态8 </suP&gt;，活化的B淋巴细胞使用糖酵解和氧化磷酸化9,10的平衡。这些研究指出调查对细胞代谢免疫细胞活化的影响的重要性。 实时的耗氧率（OCR）和细胞外酸化率（ECAR），作为氧化磷酸化和糖酵解的指标同时测量，是解决能源生产途径11-13的状态的共同战略。为了达到这个目的，一个细胞外磁通分析器，如海马XF 96，是经常使用。这种仪器能迅速在细胞类型或不同时刺激条件比较OCR和ECAR变化。迄今为止，各种细胞类型，包括免疫细胞，一直在使用这些器件的研究。但是，专门为免疫细胞设计了一个优化的协议不可用。 免疫细胞，特别是淋巴细胞，从OT不同她的细胞类型中的几个关键方面。淋巴细胞是不为体外条件下14-16较长的时间存活脆弱的细胞。这是当他们在次优生长培养基缺乏必需营养物，例如那些在细胞外通量分析中使用培养的更大的问题。与巨噬细胞和多细胞，淋巴细胞不坚持塑料表面;因此，关键是要它们附加到分析板，而不会产生应力。最后，一些淋巴细胞亚群可能是极其罕见的，并在需要的，最佳数量收获它们可能是具有挑战性的17-19。 在这里，我们提供一种专门用于淋巴细胞开发的优化的协议。使用脾细胞，B淋巴细胞和幼稚的 CD4 + T淋巴细胞从小鼠脾和淋巴结孤立节点20，我们展示自己的休息状态糖酵解和氧化磷酸化的特点，在differenT细胞密度。数据标准化为占每个中的初始细胞数的差异以及通过测量端测定细胞裂解物的蛋白质浓度，这是成正比的细胞数。我们的协议不仅提供了用于胞外磁通测定活的淋巴细胞的快速分离的准则，但它也允许在不理想的细胞浓度工作而不会损害数据质量。

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">PBS (pH 7.2)</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">20012-050</td>
			<td style="width:283px;">To make MACS buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bovine Serum Albumin BSA</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">A3803</td>
			<td style="width:283px;">To make MACS buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">0.5M EDTA, pH 8</td>
			<td style="width:139px;">Quality Biological</td>
			<td>10128-446</td>
			<td style="width:283px;">To make MACS buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">autoMACS rinsing solution</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-091-222</td>
			<td style="width:283px;">Instead of PBS + EDTA to make MS buffer. Also used in autoMACS Pro Separator.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">RPMI-1640</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">11875-093</td>
			<td style="width:283px;">Contains phenol red and L-glutamine</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">10437-028</td>
			<td style="width:283px;">For heat-inactivation, thaw frozen stock bottle in a 37 &#176;C water bath and then inactivate at 56 &#176;C for 30 minutes. Aliquot heat-inactivated serum for storage (e.g., in 50 mL conical tubes) and freeze at -20 &#176;C until needed.&#160;</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:216px;">Penicillin-Streptomycin (Pen Strep)</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">15140-122</td>
			<td style="width:283px;">Combine Pen Strep with L-Glut 1:1 (if making 500mL media, make a total of 12 mL Pen Strep/L-Glut); keep aliquots at -20 &#176;C until ready to make media.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">L-Glutamine 200mM (L-Glut)</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">25030-081</td>
			<td style="width:283px;">Component of RF10 and stress test media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sodium pyruvate 100mM</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">11360-070</td>
			<td style="width:283px;">Component of RF10 and stress test media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">HEPES 1M</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">15630-080</td>
			<td style="width:283px;">Component of RF10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">11140-050</td>
			<td style="width:283px;">Component of RF10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">2-Mercaptoethanol (55 mM)</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">21985-023</td>
			<td style="width:283px;">Component of RF10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Falcon 70 &#956;m cell strainer</td>
			<td style="width:139px;">Falcon-Fischer Scientific</td>
			<td style="width:104px;">87712</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Monoject 3 mL Syringe, Luer-Lock Tip</td>
			<td style="width:139px;">Covidien</td>
			<td style="width:104px;">8881513934</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Falcon 15mL Conical Centrifuge Tubes&#160;</td>
			<td style="width:139px;">Falcon-Fischer Scientific</td>
			<td style="width:104px;">14-959-53A</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Falcon 50mL Conical Centrifuge Tubes&#160;</td>
			<td style="width:139px;">Falcon-Fischer Scientific</td>
			<td style="width:104px;">14-432-22</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ACK Lysing Buffer</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:104px;">10-548E</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">CellTrics 30 &#956;m cell filter, sterile, single-packed</td>
			<td style="width:139px;">Partec CellTrics</td>
			<td>04-004-2326</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">B Cell Isolation Kit, mouse</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td>130-090-862</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Na&#239;ve CD4+ T cell isolation kit, mouse</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td>130-104-453</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">LS Column</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MidiMACS separator</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td style="width:283px;">Magnet for separation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MACS MultiStand</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td style="width:283px;">Holder for magnets</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">autoMACS Rinsing Solution</td>
			<td style="width:139px;"></td>
			<td>130-091-222</td>
			<td style="width:283px;">Rinsing solution for autoMACS Pro Separator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">autoMACS Pro Separator Instrument</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td style="width:104px;">N/A</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">2-Deoxy-D-glucose (2-DG)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td>D8375-5G</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Cell-Tak</td>
			<td style="width:139px;">Corning</td>
			<td style="width:104px;">354240</td>
			<td style="width:283px;">Cell adhesive. Take care to note concentration, as each lot is different (package is 1 mg).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Oligomycin</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">75351</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">2,4 Dinotrophenol (2,4-DNP)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">D198501</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Antimycin A</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">A8674</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Rotenone</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">R8875</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glucose</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">G8270</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Halt Protease Inhibitor Cocktail</td>
			<td style="width:139px;">ThermoFisher Scientific</td>
			<td style="width:104px;">78429</td>
			<td style="width:283px;">Supplied as 100X cocktail, combine with RIPA to form 1X solution for lysis.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">RIPA Buffer</td>
			<td style="width:139px;">Boston BioProducts</td>
			<td style="width:104px;">BP-115</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:139px;">ThermoFisher Scientific</td>
			<td style="width:104px;">23225</td>
			<td style="width:283px;">Follow manufacturer&#39;s instructions</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:216px;">Seahorse XFe96 FluxPak</td>
			<td style="width:139px;">Seahorse Bioscience</td>
			<td style="width:104px;">101085-004</td>
			<td style="width:283px;">Includes assay plates, cartridges, loading guides for transferring compounds to the assay cartridge, and calibrant solution.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Seahorse XF Base Medium</td>
			<td style="width:139px;">Seahorse Bioscience</td>
			<td style="width:104px;">102353-100</td>
			<td style="width:283px;">Used to prepare stress test media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Seahorse XFe96 Extracellular Flux Analyzer</td>
			<td style="width:139px;">Seahorse Bioscience</td>
			<td style="width:104px;"></td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Stericup Sterile Vacuum Filter Units, 0.22 &#956;m</td>
			<td style="width:139px;">EMD Millipore-Fisher Scientific</td>
			<td style="width:104px;">SCGPU10RE</td>
			<td style="width:283px;">Used to sterile filter media.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sodium bicarbonate</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">487031</td>
			<td style="width:283px;">Dissolved to 0.1M and used to dilute Cell-Tak.</td>
		</tr>
	</tbody>
</table>

an optimized protocol to analyze glycolysis and mitochondrial respiration in lymphocytes

淋巴细胞通过激活细胞内信号通路，这反过来又导致迅速的细胞增殖，迁移和分化，细胞因子的产生以各种刺激作出响应。所有这些事件都紧密连接到电池的能量状态，因此，研究了产生能量的途径可提供关于这些细胞的整体功能的线索。胞外通量分析仪是用于评估糖酵解和线粒体呼吸的在许多细胞类型的性能常用设备。此系统已用于研究免疫细胞在几个公开的报道，但对淋巴细胞特别优化的全面的协议是缺乏。淋巴细胞，在体外条件下存活不佳脆弱的细胞。通常情况下淋巴细胞亚群是罕见的，具有低细胞数量的工作是不可避免的。因此，需要能够解决这些困难的实验策略。在这里，我们提供了一个协议以便能够从淋巴组织可行的淋巴细胞迅速隔离，并为它们的代谢状态的细胞外流量分析仪进行分析。此外，我们提供一种在不同的细胞密度几个淋巴细胞亚型代谢活性进行比较的实验结果。这些观察表明，我们的协议可以用来甚至在低细胞浓度达到一致，以及标准化的结果，并因此可能在未来的研究集中于在免疫细胞的代谢事件的表征广泛的应用。

真核细胞利用糖酵解的集成网络，三羧酸（TCA）循环，和氧化磷酸化，以满足他们的大部分能源需求的，并为细胞生长和增殖所需的中间体。这些途径开始与无葡萄糖的葡萄糖-6-磷酸，随后被加工成丙酮酸的形式细胞内俘获。丙酮酸要么降低为乳酸或转运到线粒体，在那里形成乙酰辅酶A（辅酶A）。乙酰辅酶A，然后进入TCA循环。 TCA循环的高能量中间体推进在电子传递链（ETC），这反过来又排出的 H +从线粒体基质，以产生跨线粒体内膜一个H +的梯度电子的运动。氧气作为最终电子受体和H +通过在F O / F &lt;返回到线粒体基质子&gt; 1复合物，其中，其潜在的能量被用于产生ATP（ 图1A）。 胞外通量测定法的工作原理依赖于在特定点与糖酵解和氧化磷酸化干扰，并评估得到的效果。为了这个目的，我们使用了葡萄糖传播中饥饿细胞糖酵解和2-脱氧葡萄糖（2-DG），将其转化为2-脱氧葡萄糖-6-磷酸，葡糖磷酸异构酶23的竞争性抑制剂，通过己糖激酶，以阻止糖酵解。鱼藤酮（ETC的一个复杂的特定I抑制剂），抗霉素A（复杂ETC的第三特异性抑制剂），寡霉素（抑制剂ATP合酶24），并且解偶联剂2,4- dinotrophenol（DNP） 有25人用来干预，以电子传递，质子梯度和ATP合成（ 图1A）相关的特定事件。代替2,4-DNP，羰CYA德纳-4-（三氟甲氧基）苯腙（FCCP）也可以使用，但可能需要进一步优化。在这两种情况下，解偶联剂应当始终滴定为特定的细胞类型在使用前，以确定所需的最佳浓度。 酵解应力测试（ 图1B）开始与在先前饥饿细胞外酸化率（ECAR）的基线测量。因为这些细胞是在它们的基部最小，因此可以实际上被看作非糖酵解，在这一点上测得的ECAR被称为非糖酵解酸化。此酸化可能对应于TCA循环产生的呼吸的 CO 2转化为HCO 3 -和H +。这之后是葡萄糖注射激活糖酵解，其作为增加细胞外酸化呈现由于乳酸的形成。这增加代表糖酵解的正常率。然后将细胞与注射寡的，其中块的ATP通过氧化磷酸化产生挑战。细胞在ATP产生这种显着的下降响应通过激活糖酵解到其最大水平，并且导致在ECAR水平（糖酵解储备）仲增加。测试是通过使用葡萄糖类似物2-DG，返回ECAR其非糖酵解水平糖酵解总抑制终止。有趣的是，已经提出的是，违背制造商的建议，最大糖酵解不必由的寡26注射实现。在具有高的糖酵解能力的细胞，如果没有在ATP的需求显著增加，糖酵解可能是完全能够应付线粒体ATP的损耗内无需是上调。与寡糖酵解率可以通过增加呼吸抑制剂被超越如鱼藤酮和myxothiazol，它提供对寡几个优点:1）它们增加ATP的需求，因为它们会导致的ATP合酶的反转，与ATP水解，泵质子以试图恢复线粒体膜电位26; 2）它们可以防止介质，它可以混淆ECAR结果（见下文）的呼吸酸化。其他方法来增加ATP需求包括添加，通过质膜ATP酶26刺激ATP水解化合物。所有这一切都应该仔细研究人员计划糖酵解压力测试时，必须考虑。 线粒体应力测试（ 图1C）开始在非饥饿细胞中的氧消耗速率（OCR）的基线测量。这之后是注射寡的，其抑制质子通过在F O / F 1络合物的返回，从而迅速hyperpolarizES线粒体膜。超极化阻止进一步质子通过呼吸道络合物抽水，以及呼吸率降低。剩余的呼吸被称为质子泄漏，它代表质子通过脂质或其他通道的流动。此超极化状态迅速通过加入偶联试剂2,4-DNP，其充当质子离子载体的逆转。在响应中，细胞尝试通过提高电子传输速率至最大回收妄图膜电位，而这又增加了OCR。最后，增加了两个ETC抑制剂（抗霉素A和鱼藤酮）的，线粒体呼吸完全停止和OCR降低到最低水平。在这个水平上，耗氧量不因线粒体活性（非线粒体）。由这些抑制剂所产生在OCR的差称为最大线粒体呼吸，这是基线呼吸和备用容量的总和。 <p类="jove_content"FO:保together.within页="1"&gt; B和T细胞隔离获得高度可行的纯淋巴细胞群。 B细胞和幼稚的 CD4 + T细胞的分离如在协议的第4节中概述，和脾细胞简单地通过如在3.3节中描述裂解红血细胞获得的。 细胞类型特异性代谢测定的灵敏度取决于生存能力和起始细胞群的纯度。因此，为了验证的分离的小鼠的T细胞，脾细胞，和B细胞和T细胞和B细胞的纯度活力，细胞的小等分试样染色流式细胞术分析（ 图2）。在正向散射面积与侧散射区域（FSC-A与SSC-A）的情节，淋巴细胞门，而这个门，向前分散唤起注意沿对角线的人群中T（FSC-H）与FSC-A地块被确定为单线。内的单峰人口，生存能力被选通，对于活/死标记（ 图2A）染色阴性的细胞中测量; T细胞存活率为97.9％，脾细胞存活率为92％，和B细胞活力为94％。 B细胞的纯度，如通过B220 + CD19 +群体26测定，为99％，而CD4 + T细胞的纯度，如通过CD44的测量-的CD4 +群体27，为98.3％（ 图2B）。 所述细胞裂解物的蛋白质浓度可以用作电镀细胞数的直接指标。 分离的淋巴细胞和脾细胞中的粘合剂涂覆的96孔测定板以5×10 5个细胞/孔，2.5×10 5细胞/孔，1.25×10 5个细胞/孔，并0.625×10 <sup&gt; 5细胞/孔。在三种细胞类型的各电镀密度合流是在光学显微镜（ 图3A）下观察。正如预期的，汇合与初始电镀密度相关。在细胞外通量分析完成后，镀裂解细胞并使用BCA测定其蛋白质浓度进行定量。对于所有类型的细胞，被证明裂解物的蛋白质浓度与初始接种密度（ 图3B），这证实裂解物的蛋白质浓度可以解释胞外通量数据时用作细胞数的正常化的精确量度是线性相关。在这个实验中使用的电镀密度已为幼稚，未刺激的淋巴细胞进行了优化。如果刺激或预先培养的细胞中使用时，可能需要进一步优化。 线粒体和糖酵解stres小号测定法依赖于镀层细胞数目。 OCR的是为在细胞外磁通分析器每种细胞类型和铺板密度测定。正如预期的那样，更高的细胞数具有更高的测量的OCR，以及更剧烈的反应，寡，2,4- DNP和抗霉素A /鱼藤酮（ 图4A）。标准化的OCR测量每个样品的蛋白浓度显示，在一般情况下，较大的细胞的数量导致更准确的OCR测量。在5和2.5×10 5细胞镀/孔导致的T细胞和脾细胞，和5和1.25×10 5细胞类似标准化的OCR测量/孔导致B细胞（ 图4B）类似标准化的OCR测量。在规范化的B细胞的细微差别的OCR测量可能是一个间接指示B细胞/相比其他细胞密度以及执行以2.5×10 5细胞更好。通过各种措施，0.625×10 5个细胞/孔，得到次优的结果，这表明该电镀密度是不够的。基线呼吸，质子泄漏，最大线粒体呼吸，非线粒体呼吸，和ATP生产对所有类型的细胞（ 图4C）电镀密度线性相关。另外，大多数的基线呼吸的用于向合成的ATP，通过在三种细胞类型中低质子泄漏所指示的。而线粒体应力实验的主要重点是测量在OCR变化，ECAR仍然是有用的记录，以确保该试验成功地进行了。类似于OCR，两个较高电镀密度导致更高ECAR和更剧烈的反应，寡，2,4- DNP和抗霉素A /鱼藤酮（ 图4D）。在在加入2,4-DNP的ECAR，和抗霉素A /鱼藤酮的剧烈变化，可能是由于除了以g变化呼吸变化lycolysis，由于CO在TCA循环中产生2被转换为HCO 3 -和H +。这个问题已被最近处理，并且存在一种简单的方法使用氧的消耗数据校正的总细胞外酸化信号，以获得真实糖酵解速率12,29。 酵解应力测定法是最成功的在最高铺板密度（ 图5A，B）。在所有类型的细胞中，5×10 5个细胞/孔的样品具有在ECAR变化最大的添加葡萄糖后，寡霉素，和2-DG（ 图5B）。此外，归一到蛋白质浓度表明，对于所有类型的细胞中，5×10 5个细胞/孔的样品，得到最佳的结果，而较低的浓度，尤其是0.625×10 5个细胞/孔，并没有显示健壮糖酵解储备能力。非glycolytiÇ酸化，糖酵解和视糖酵解容量线性对所有类型的细胞（ 图5C）电镀密度相关。为糖酵解应力实验中，OCR图表明葡萄糖略有刺激线粒体呼吸，然后由寡治疗抑制;在加入2-DG的没有影响的OCR（ 图5D）。 OCR的图形可以用作该糖酵解应力实验成功地进行了进一步的指示器。此外，OCR图可以用来校正ECAR图，如果需要的话，如在线粒体应力测试12,29的情况下，如上所述。 <img alt="图1" src="/files/ftp_upload/54918/54918fig1.jpg" /> 图 1:糖酵解（左）的胞外通量测定纲要 （A）插图和氧化磷酸化（右）展示的作用在细胞外通量测定法中使用的代谢药物。 （B）的胞外酸化率（ECAR）图的示意图;耗氧率（OCR）图（C）的示意图。 <a href="http://ecsource.jove.com/files/ftp_upload/54918/54918fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54918/54918fig2.jpg" /> 图2:T细胞，B细胞和脾细胞来确定存活和纯度的逐步浇注流式细胞曲线示出的T细胞，脾细胞，和B细胞的生存力（A）;和T细胞和B细胞（B）的纯度。结果代表至少有三个独立的实验。 <a href="http://ecsource.jove.com/files/ftp_upload/54918/54918fig2large.jpg" target="_blank">请点击此处查看次的放大版本</a>是图。 <img alt="图3" src="/files/ftp_upload/54918/54918fig3.jpg" /> 图3: 细胞汇合，在不同的电镀密度每种细胞类型的溶胞产物的蛋白浓度相关 的（A）测定板孔在电镀密度细胞光显微照片，从5×10 5个细胞/孔至0.625×10 5个细胞/好。比例尺表示50微米。 （B）的溶胞产物的蛋白浓度在不同的电镀密度，如通过BCA测定法测量。结果代表至少有三个独立的实验。 <a href="http://ecsource.jove.com/files/ftp_upload/54918/54918fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54918/54918fig4.jpg" /> 图4:<html> 线粒体应力测定。原料（A）和标准化的（B）中的OCR对于每个细胞类型和铺板密度被示出。 （C）在基线，最大线粒体和非线粒体呼吸的水平，以及连接于质子泄漏或ATP的产生氧消耗细胞依赖性数的变化，示出了对于每个细胞类型。从线粒体应力试验获得（D）的原始ECAR值显示。每个数据点代表7-8井的标准偏差的平均值。标记的箭头表示寡注射（O），2,4-DNP（D），鱼藤酮和/抗霉素A（R / A）。结果代表至少有三个独立的实验。 <a href="http://ecsource.jove.com/files/ftp_upload/54918/54918fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> _upload / 54918 / 54918fig5.jpg"/&gt; 图 5: 糖酵解应力测定原料（A）和标准化的（B）的ECAR每种细胞类型和铺板密度被示出。 （ 三 ）ECAR非糖酵解酸化，葡萄糖诱导的糖酵解和总糖酵解产能细胞依赖性数变化显示。从糖酵解应力试验获得（D）的原料的OCR的数值。每个数据点代表7-8井的标准偏差的平均值。标记的箭头表示葡萄糖（G），寡（O）和2-脱氧葡萄糖（2-DG）注射。结果代表至少有三个独立的实验。 <a href="http://ecsource.jove.com/files/ftp_upload/54918/54918fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes at JoVE.com

分析淋巴细胞糖酵解和线粒体呼吸的优化方案免疫学和感染|视频

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

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29.5K 次观看.  National Institutes of Health. The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

视频: An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

科研

免疫与感染

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

全细胞MALDI-TOF质谱法是一种准确，快速的方法来分析巨噬细胞活化的不同模式

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

流量Virometry来分析病毒颗粒的抗原谱和外囊泡

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

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 ...

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

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