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><tbody><tr><td>PBS (pH 7.2)</td><td>Gibco-ThermoFisher</td><td>20012-050</td><td>To make MACS buffer</td></tr><tr><td>Bovine Serum Albumin BSA</td><td>Sigma-Aldrich</td><td>A3803</td><td>To make MACS buffer</td></tr><tr><td>0.5 M EDTA, pH 8</td><td>Quality Biological</td><td>10128-446</td><td>To make MACS buffer</td></tr><tr><td>autoMACS rinsing solution</td><td>Miltenyi Biotec</td><td>130-091-222</td><td>Instead of PBS + EDTA to make MS buffer. Also used in autoMACS Pro Separator.</td></tr><tr><td>RPMI-1640</td><td>Gibco-ThermoFisher</td><td>11875-093</td><td>Contains phenol red and L-glutamine</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco-ThermoFisher</td><td>10437-028</td><td>For heat-inactivation, thaw frozen stock bottle in a 37 &amp;deg;C water bath and then inactivate at 56 &amp;deg;C for 30 min. Aliquot heat-inactivated serum for storage (&lt;em&gt;e.g.&lt;/em&gt;, in 50 ml conical tubes) and freeze at -20 &amp;deg;C until needed.&amp;nbsp;</td></tr><tr><td>Penicillin-Streptomycin (Pen Strep)</td><td>Gibco-ThermoFisher</td><td>15140-122</td><td>Combine Pen Strep with L-Glut 1:1 (if making 500 ml media, make a total of 12 ml Pen Strep/L-Glut); keep aliquots at -20 &amp;deg;C until ready to make media.</td></tr><tr><td>L-Glutamine 200 mM (L-Glut)</td><td>Gibco-ThermoFisher</td><td>25030-081</td><td>Component of RF10 and stress test media</td></tr><tr><td>Sodium pyruvate 100 mM</td><td>Gibco-ThermoFisher</td><td>11360-070</td><td>Component of RF10 and stress test media</td></tr><tr><td>HEPES 1 M</td><td>Gibco-ThermoFisher</td><td>15630-080</td><td>Component of RF10</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)</td><td>Gibco-ThermoFisher</td><td>11140-050</td><td>Component of RF10</td></tr><tr><td>2-Mercaptoethanol (55 mM)</td><td>Gibco-ThermoFisher</td><td>21985-023</td><td>Component of RF10</td></tr><tr><td>Falcon 70 &amp;mu;m cell strainer</td><td>Falcon-Fischer Scientific</td><td>87712</td><td>Used in cell isolation</td></tr><tr><td>Monoject 3 ml Syringe, Luer-Lock Tip</td><td>Covidien</td><td>8881513934</td><td>Used in cell isolation</td></tr><tr><td>Falcon 15 ml Conical Centrifuge Tubes&amp;nbsp;</td><td>Falcon-Fischer Scientific</td><td>14-959-53A</td><td>Used in cell isolation</td></tr><tr><td>Falcon 50 ml Conical Centrifuge Tubes&amp;nbsp;</td><td>Falcon-Fischer Scientific</td><td>14-432-22</td><td>Used in cell isolation</td></tr><tr><td>ACK Lysing Buffer</td><td>Lonza</td><td>10-548E</td><td>Used in cell isolation</td></tr><tr><td>CellTrics 30 &amp;mu;m cell filter, sterile, single-packed</td><td>Partec CellTrics</td><td>04-004-2326</td><td>Used in cell isolation</td></tr><tr><td>B Cell Isolation Kit, mouse</td><td>Miltenyi Biotec</td><td>130-090-862</td><td>Used in cell isolation</td></tr><tr><td>Na&amp;iuml;ve CD4&lt;sup&gt;+&lt;/sup&gt; T cell isolation kit, mouse</td><td>Miltenyi Biotec</td><td>130-104-453</td><td>Used in cell isolation</td></tr><tr><td>LS Column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>Used in cell isolation</td></tr><tr><td>MidiMACS separator</td><td>Miltenyi Biotec</td><td>130-042-302</td><td>Magnet for separation</td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td>Holder for magnets</td></tr><tr><td>autoMACS Rinsing Solution</td><td></td><td>130-091-222</td><td>Rinsing solution for autoMACS Pro Separator</td></tr><tr><td>autoMACS Pro Separator Instrument</td><td>Miltenyi Biotec</td><td>N/A</td><td></td></tr><tr><td>2-Deoxy-D-glucose (2-DG)</td><td>Sigma-Aldrich</td><td>D8375-5G</td><td></td></tr><tr><td>Cell-Tak</td><td>Corning</td><td>354240</td><td>Cell adhesive. Take care to note concentration, as each lot is different (package is 1 mg).</td></tr><tr><td>Oligomycin</td><td>Sigma-Aldrich</td><td>75351</td><td></td></tr><tr><td>2,4-Dinotrophenol (2,4-DNP)</td><td>Sigma-Aldrich</td><td>D198501</td><td></td></tr><tr><td>Antimycin A</td><td>Sigma-Aldrich</td><td>A8674</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Halt Protease Inhibitor Cocktail</td><td>ThermoFisher Scientific</td><td>78429</td><td>Supplied as 100x cocktail, combine with RIPA to form 1x solution for lysis.</td></tr><tr><td>RIPA Buffer</td><td>Boston BioProducts</td><td>BP-115</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>ThermoFisher Scientific</td><td>23225</td><td>Follow manufacturer&#039;s instructions</td></tr><tr><td>Seahorse XFe96 FluxPak</td><td>Seahorse Bioscience</td><td>101085-004</td><td>Includes assay plates, cartridges, loading guides for transferring compounds to the assay cartridge, and calibrant solution.</td></tr><tr><td>Seahorse XF Base Medium</td><td>Seahorse Bioscience</td><td>102353-100</td><td>Used to prepare stress test media</td></tr><tr><td>Seahorse XFe96 Extracellular Flux Analyzer</td><td>Seahorse Bioscience</td><td></td><td></td></tr><tr><td>Stericup Sterile Vacuum Filter Units, 0.22 &amp;mu;m</td><td>EMD Millipore-Fisher Scientific</td><td>SCGPU10RE</td><td>Used to sterile filter media.</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>487031</td><td>Dissolved to 0.1 M 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

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

造血干细胞代谢分析

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

科研

发育生物学

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

免疫与感染

Measuring Yeast Mitochondrial Respiration While Maintaining Cellular Context

Mitochondrial Respiration Quantification in Yeast Whole Cells

Metabolism is mainly coordinated by cellular energy availability and environmental conditions. Assays for knowing how cells adapt energetic metabolism to different nutritional and environmental conditions give valuable information to elucidate molecular mechanisms. Oxidative phosphorylation is the primary source of ATP in most cells, and mitochondrial respiration activity is a key component of oxidative phosphorylation, maintaining mitochondrial membrane potential for ATP synthesis. Mitochondrial respiration is often studied in isolated mitochondria that are missing the cellular context. Here, we present a method for quantifying mitochondrial respiration in yeast-intact cells. This method applies to any yeast species, although it has been generally used for Saccharomyces cerevisiae cells. First, the yeast growth in specific conditions is tested. Then, cells are washed and resuspended in deionized water with a 1:1 ratio (w/v). Cells are then placed in an oximeter chamber with constant stirring, and a Clark electrode is used to quantify oxygen consumption. Some molecules are sequentially placed into the chamber and selected according to this effect on the electron transport chain or ATP synthesis. ATPase inhibitor oligomycin is first added to measure respiration coupled to ATP synthesis. Afterward, an uncoupler is used to measure the maximal respiratory capacity. Finally, a mix of electron transport chain inhibitors is added to discard oxygen consumption unrelated to mitochondrial respiration. Data are analyzed using a linear regression to obtain the slope, representing the oxygen consumption rate. The advantage of this method is that it is specific for yeast mitochondrial respiration, maintaining the cellular context. It is essential to highlight that inhibitors used in mitochondrial respiration quantification could vary between yeast species.

Mitochondrial respiration in yeast whole cells is a valuable indicator of cell bioenergetics. Here, we present a protocol to quantify this phenotype applicable to different yeast species.

Metabolism is mainly coordinated by cellular energy availability and environmental conditions. Assays for knowing how cells adapt energetic metabolism to ...

生物化学

Analysis of Human Natural Killer Cell Metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

人类自然杀手细胞代谢分析

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;s defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.
This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.
In this paper, we polarized the na&#239;ve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.

实时监测细胞因子分化人原代T细胞中的线粒体呼吸

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is ...

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

实时测量中性粒细胞的线粒体生物能量特征

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

生物学

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.
Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 &#215; 105 cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

分析脾和心脏巨噬细胞以及骨髓单核细胞中的 离体 代谢通量

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, ...

Measuring Mitochondrial Function of Na&#239;ve and Effector CD8 T Cells

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. Lymphocyte biology has been explored using bioenergetic analysis and has now become a critically import tool in the field. Thus, we sought to optimize a bioenergetic analysis assay that can be adapted with pretreatments and acute injection for receptor stimulations. Here, we evaluated CD8 T cell ex vivo metabolism using the Cell Mito Stress Test to assess rates of oxygen consumption and extracellular acidification in na&#239;ve and effector CD8 T cells. Antigen-specific effector CD8 T cells were derived via ex vivo stimulation, and na&#239;ve CD8 T cells harvested from splenocytes and isolated with magnetic bead column separation. 
Pretreatments are performed in microplates and we detail how to prepare sensor cartridges. We show how injection ports can loaded with drugs to indirectly measure metabolic capacities and with metabolic modulators, this protocol can be used to study specific enzyme activity. T-cell receptor stimulations can be studied in real time with acute injection and stimulation with anti-CD3/CD28 using the injection ports. Instrument analyzers are used for measurements and data collection and data visualization is done with software programs to interpret cellular metabolism. This strategy produces an extensive amount of data on immune cell biology and mitochondrial bioenergetics allowing researchers to customize the protocol in numerous ways to explore CD8 T cell metabolism.

CD8 T cell bioenergetics can be interrogated using the Mito Stress Test. This methodology can be used to study acute and chronic metabolic programming. This protocol describes approaches to examine the relationships between T cell receptor biology and bioenergetic analysis.

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

摘要

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造血干细胞代谢分析

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Mitochondrial Respiration Quantification in Yeast Whole Cells

人类自然杀手细胞代谢分析

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

实时监测细胞因子分化人原代T细胞中的线粒体呼吸

实时测量中性粒细胞的线粒体生物能量特征

隔离和线粒体从培养细胞和小鼠组织功能分析

分析脾和心脏巨噬细胞以及骨髓单核细胞中的离体代谢通量

测量初始和效应 CD8 T 细胞的线粒体功能

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

摘要

探索更多视频

此视频中的章节

造血干细胞代谢分析

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Mitochondrial Respiration Quantification in Yeast Whole Cells

人类自然杀手细胞代谢分析

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

实时监测细胞因子分化人原代T细胞中的线粒体呼吸

实时测量中性粒细胞的线粒体生物能量特征

隔离和线粒体从培养细胞和小鼠组织功能分析

分析脾和心脏巨噬细胞以及骨髓单核细胞中的 离体 代谢通量

测量初始和效应 CD8 T 细胞的线粒体功能

分析脾和心脏巨噬细胞以及骨髓单核细胞中的离体代谢通量