The authors thank Epithelix Sarl and Biopredic International for providing the airway cells and hepatic cells micrographies and Neil Smith for the figure illustrating the experimental procedure.

<html>注:代谢活性测定的一般工作流程如图2所列。这个工作流程的每一步是随后在接下来的段落中详细说明。试剂和设备的详细表格中材料的表中给出。 <img alt="图2" src="/files/ftp_upload/55502/55502fig2.jpg" /> 图2:工作流中的代谢探针检测。将细胞接种并生长到足够密度。甲CYP诱导步骤可能依赖于所测定的酶活性是必需的。探针底物和抑制剂添加至细胞培养物。温育期后，将培养基收集用于分析和裂解细胞用于蛋白质定量。该介质处理以用于通过质谱法或发光测定的探针基板的代谢产物的分析。广告/ 55502 / 55502fig2large.jpg"目标=‘_空白’&gt;点击此处查看该图的放大版本。 1.细胞培养<img alt="图3" src="/files/ftp_upload/55502/55502fig3.jpg" /> 图3:肝细胞系培养物的光透射显微照相。在这个协议5中所述的细胞系通常分化与培养胆管和肝细胞之间的50％的分割（100×）。比例尺=100μm左右。 <a href="http://ecsource.jove.com/files/ftp_upload/55502/55502fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li> 肝细胞株培养 注:将市售的肝细胞系用作代谢感受态细胞的一个例子。该细胞系从由病毒感染引起7肝癌衍生。在融合时，该细胞系分化为cholangiocyte-和肝细胞样细胞（ 图3），并且进一步的细节可以在Guillouzo 7中找到。 2％DMS​​O的向培养基中包含支持多种的CYP的该细胞系和表达的分化。 <ol><li>从液氮中取出与细胞低温小瓶中，并放置在运输干冰。转移到冷冻管的水浴预热至37℃1-2分钟，并在层流罩放置之前消毒用70％乙醇小瓶的外部。 </li><li>虽然在无菌条件下工作时，小心地打开低温小瓶以释放任何多余的压力和低温小瓶中的内容吸取到10ml管中在37℃下填充有9mL的预温 - 威廉姆斯E培养基。 </li><li>离心溶液10分钟，在室温下400×g离心，弃去上清液，并将细胞重新悬浮于5mL的专有的解冻介质预加热，在37℃，补充有谷氨酰胺或谷氨酰胺补充物（2mM的终浓度）（Williams的E +补充谷氨酰胺+解冻补充剂）。 </li><li>使用500微升等分试样用细胞计数器或任何其他合适的细胞计数技术计数活细胞。 </li><li>种子24孔胶原包被的板中的细胞以0.6×10 6个活细胞/孔的密度在0.5毫升解冻介质的最终体积。 </li><li>放置在培养箱中的板（S）在37℃下用5％/ 95％CO 2 /空气和100％相对湿度。 </li><li>接种细胞24个小时后，更换用0.5ml预热维护/代谢工作介质的（Williams的E +补充谷氨酰胺+代谢补充物（该培养基已包含DMSO））的平台。改变介质每两天。 </li><li>在培养数天的细胞形成一个亚汇合骨小梁结构（ 图3）。最佳代谢活性7天和10之间通常观察到，并且在第4天最低。 </li></ol></li></ol><img alt="图4" src="/files/ftp_upload/55502/55502fig4.jpg" /> 图4:在空气-液体界面8生长爱茜蓝染色呼吸道细胞的截面图。 纤毛，粘液生产和基底细胞清晰可见。比例尺=250μm左右。 <ol start="2"><li> 差异化的呼吸道上皮细胞培养 注:该协议是示出在气-液界面上的插入件8（ 图4）生长的市售完全分化的主气道上皮细胞。组织培养保留了粘膜纤毛表型和细胞极性8。细胞可以是鼻或支气管来源的。在这里，用人类的鼻腔细胞。将细胞在24培养板格式传送插入在空气 - 液体界面。为了便于运输和避免介质溢出插入件随嵌入在用培养基混合琼脂糖基质基底部分。 <ol><li>在递送单元中，在一个干净的层流罩放置之前消毒含有24将用70％乙醇溶液将板。 </li><li>通过加入预热的700μL专有培养基制备的无菌24孔板中。 </li><li>使用无菌镊子轻轻从琼脂糖基质撞出所述插入单元插入转移到新的板预先加载平台。 </li><li>放置在培养箱中的板（S）在37℃下用5％/ 95％CO 2 /空气和100％相对湿度。 </li><li>每2天插入物转移到新的无菌24孔板预装载有700μL的暖气道专有细胞培养基。 </li><li>评估气道组织的使用方法，如经上皮电阻（TEER），或完整性纤毛使用装有实时视频成像系统9的显微镜拍频（CBF）。 TEER和CBF的测量在10 Kuehn的描述。 </li></ol></li></ol> 2. CYP活性定量在细胞培养物注意:作为一般规则，优选溶剂如DMSO和甲醇可用于在本文所提出的不同的代谢探针和抑制剂制备储备溶液。然而，建议在培养基中的最终DMSO浓度保持在最低水平（1％或更低），因为高浓度的能抑制代谢酶。如果可能的话，还建议使用无酚红和无血清培养基中与代谢探针的温育过程中，因为酚红可以用的CYP和II期酶的活性和血清组分如白蛋白可降低探针的可用性干涉。它并不总是能够严格遵守这些<html>准则。例如，呼吸道细胞培养基是专有的，含有酚红。专有肝细胞系的代谢平台7，因为它在细胞采取肝细胞样表型需要已经包含高于1％DMSO。 注:最后，在这里，术语特异性探针/抑制剂使用，但这个术语有慎重考虑。事实上，这是非常罕见的代谢探针是酶底物排斥。这里所描述的探针，然而，对于它们的目标酶的高亲和性，并因此被认为是活性的可靠标记。 <ol><li> 代谢探针，诱导物，和抑制剂 <ol><li>使用基于质谱法探头 注:在本节描述的探针和抑制剂是合适的，以测试CYP2As 11的活性，CYP1A2 12，REF"&gt; 13，CYP2B6 7，14，CYP2F1 15，图 16和CYP2E1 17由UPLC-MS分析。探针，抑制剂和相应CYP在表1中具有相应的参考文献中描述。 表1还列出了CYP的代谢产物这是通过质谱法进行定量的活性。所有涉及组织培养和解决方案的组织培养中的工作是在一个无菌组织培养橱中进行。储备溶液可以在DMSO中的所有探针底物来制备。 <ol><li>在实验之前，只对代谢探针准备两个串联电池单元的插入孔或插孔中，使用一个系列的用于抑制实验，其他。制备的第三系列的细胞作为介质空白对照。它们可以是在单独的板或在用最少的三次重复的相同平板上。板的一例铺设出被呈现在图5中。 </li><li>在实验的当天，代替以新鲜培养基的450微升的细胞培养基。 </li><li>制备在无菌罩10倍浓度使用细胞培养基通过以下方法解决。混合10×CYP探针，10X CYP抑制剂，和10倍的CYP抑制剂加10X探针。 注:抑制剂和探针的硕士储备溶液可以在100％DMSO来制备（ 例如 ，1000倍的股票），并进一步在培养基中稀释到，以避免超过1％的细胞培养物中的最终DMSO浓度获得10倍的溶液。其旨在用于与细胞孵育的最后1X浓度示于表1中 。 </li><li>过滤用0.2μm注射器过滤器不同10X探针溶液。 </li><li>添加培养基的50μL含有10×CYP抑制剂与系列为抑制实验和预温育30分钟而制备的细胞。 </li><li> - [RE放置细胞的培养基中的抑制系列用新鲜培养基的450μL，并添加含有10×CYP抑制剂和10X探针基底介质的50μL。 </li><li>添加到其它单元与10倍的探针基底介质的50μL，并添加在培养基中稀释（ 例如 ，DMSO）到空白细胞对照车辆的等效量， 如图5。 </li><li>孵育细胞0 - 5分钟（时间0，背景对照），并在细胞培养箱16小时。 注:此孵育时间与呼吸道细胞和肝细胞系成功地使用;然而新鲜原代肝细胞具有较高的代谢能力，所述细胞制剂的质量和供体的遗传组成，因此较短的时间未决（ 例如 ，2〜4小时）可能是合适的。它总是建议测试不同的细胞系或细胞类型时执行的时间过程实验。 </li><li>在潜伏期，科尔结束ECT在用于探针的代谢产物的定量单独标记的管的平台。冻结将来的处理中。 </li><li>裂解细胞的蛋白定量。 注意:任何标准的蛋白定量试剂盒和协议适合于该步骤。 BCA是一个合适的选择。这个步骤是可选的，如果细胞在另一个试验中使用。 </li></ol></li><li> 媒体处理 注:从孵化阶段收集到的介质包含父探测化学以及它的代谢产物。要衡量一个特定CYP活性只有感兴趣的CYP催化反应的产品应该被量化。阶段I代谢产物通常与II期代谢（缀合）串联处理，因此不能作为这样来检测。因此，为了获得一个CYP活性的最精确的定量，一个去缀合步骤是必需的，以消除任何二期代谢修改。由于第一阶段的共轭我代谢产物通常涉及葡糖醛酸化或硫酸化，去缀合是通过处理的样品的由葡糖醛酸酶实现和硫酸酯酶18。葡萄糖醛酸酶和硫酸酯酶活性是在pH 4.5-5.0和硫酸酯酶活性是在pH 6.0-6.5最高正在实现pH依赖性与最高葡萄糖醛酸酶的活性。因此，此步骤需要调节pH。在这里所描述的方法中，我们使用pH条出于实际的原因，因为大多数pH计探针不适合在1.5mL微离心管中测量的pH值。可商购的pH值条可以以低到0.3至0.2个pH单位的分辨率被发现。 <ol><li>转移温育培养基的250微升到1.5mL微管中，并添加4-甲基伞形酮内标储备溶液以达到100nM的最终浓度。 </li><li>通过添加1M盐酸调节培养基的pH至4.5-5.0。添加盐酸的1μL的时间和pH值验证通过吸取2μL培养基到pH试纸。达到5.0的pH值通常需要不超过5-10μL的1M HCl的更多。 </li><li>添加从罗马蜗牛 β葡萄糖醛酸酶/芳基硫酸酯酶的150个单位（1.8μL的85000单位/ mL储备的），并在37℃下孵育1个小时。通过加入冷的甲醇的250μL终止反应（-20℃）。在-20℃或过程进一步存储样本。 </li><li>使用真空离心机在45℃下蒸发溶剂。水溶液:用500微升30％乙腈的重构样本。将样品准备用于质谱分析，并且可以存储在安伯·格拉斯小瓶中-20℃。 </li></ol></li><li> 基于质谱法量化 注:所有的UPLC和质谱仪设置在专用参考表1详细。对于定量分析，建立校准曲线是必需的。卡利bration曲线是通过运行的化合物的公知的稀释范围进行量化获得。在这种情况下，经10个不同浓度（ 表2），其中至少6的应该是线性范围19的20％之内所产生的校正曲线。检测的最低水平被确定为给出一个信号大于或等于3倍的平均背景信号的最低浓度。定量的最低水平被确定为最低浓度的其给出信号大于或等于10倍两者都确定在至少3个与一式三份样品独立实验的平均背景信号的样本。 <ol><li>根据表2制备在组织培养基中的合成代谢产物的连续稀释液，包括内部标准。的每标准稀释250μL的最终体积是足够的。 </li><li>蒸发的标准系列稀释液作为第一描述EP 2.1.2.4，重悬在30％乙腈的体积:相当于水介质之前蒸发的体积（ 例如 ，250微升30％乙腈:水如果起始标准溶液为介质的250微升）。 </li><li>添加每个连续稀释的标准的250μL成琥珀色UPLC玻璃小瓶用于注射在UPLC-MS自动进样器。 </li><li>添加测试样品（250微升），以琥珀色UPLC小瓶，并放置在自动进样器UPLC。 </li><li>随机所有的小瓶。 注意:我们使用的质谱仪配置是耦合到UPLC在补充表1详细混合三重四极/线性离子阱质谱仪。其它平台会有不同的软件和配置，但会遵循类似的步骤来执行量化。 </li><li>运行使用补充的表1视具体探针量化详述的设置UPLC-MS。 </li><li>使用了"定量-建立定量方法"，在质谱仪定量工具工具（ 材料的表 ），以生成从所取得的合成标准和内标校准曲线。 </li><li>使用"定量向导"选择样品批次和样品量化和执行"定量方法"。质谱仪定量软件显示与每个测试样品中的目标化合物的定量值的电子表格。 </li></ol></li><li> 使用基于荧光探针方法 注意:发光的探针来测量一系列CYP活动的可商购获得;然而，并不是所有的都适合基于细胞的测定。在这里，被呈现给测量CYP1A1 / CYP1B1活性的协议。 CYP1A1 / 1B1被选择来说明如何测定诱导的CYP的活性和得到的如何发光的探针可用于作为ALTER一个例子原产于质谱为基础的方法。 表3总结了我们用于气道细胞和肝细胞系中的浓度和温育时间;但是，那些可能需要适用于其他类型的细胞。其他发光的探针是可商购的，并且可以用作用于各种的CYP的基材。 <ol><li>准备足够的细胞以包括非诱导控制，感应测试，和感应+抑制剂试验。板布局的一个例子示于图5B。 </li><li>为了诱导CYP1A1 / 1B1，孵育所述细胞用10nM的TCDD在介质一段72个小时的最终浓度（24至48小时较短的时间也可以是合适的）。 注意:二恶英是一种致癌物质和致畸剂。穿戴适当的防护设备，检查供应商材料安全数据表，并在使用前进行风险评估。 </li><li>在此之后，除去介质，用PBS冲洗3次井，并添加一些新鲜的培养基。 </li><li>镨IOR到温育与发光的报告探针的细胞，预孵育诱导的细胞的子集指定为30分钟，用10μMCYP1A1 / 1B1抑制剂，α萘黄酮的终浓度，在平台。 </li><li>在50μM的终浓度添加发光的探针LUC-CEE（荧光素6&#39;-氯乙基醚），以使在非诱导，诱导和诱导的+抑制剂的细胞。 </li><li>孵育在细胞培养箱中4个小时。在孵育时间结束时收集的发光的探针定量的媒介。 </li><li>转移25μL培养基至白色不透明96孔板中，并添加由制造商提供的荧光素检测试剂的25μL。孵育在室温下20分钟。读板立即用光度计。 </li><li>用PBS冲洗细胞并裂解总蛋白定量。 注意:这一步是可选的，如果活细胞被保留为将来的实验。一世t是重要然而需要注意的是诱导例如TCDD是有毒的，会损害细胞。 </li></ol></li></ol></li></ol><img alt="表格1" src="/files/ftp_upload/55502/55502table1.jpg" /> 表1: 与他们相应的酶代谢探针，产品和抑制剂的名单。分子量，并与气道和肝细胞中使用推荐的浓度也表示。 <a href="http://ecsource.jove.com/files/ftp_upload/55502/55502table1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55502/55502fig5.jpg" /> 图5:建议的24孔平板布局来执行CYP测定。 （A），用于组成型表达的CYP的推荐布局包括用于空白介质的一系列孔，代谢探针，和代谢探针加抑制剂。该布局可通过正在测试的时间点的数量相乘。 （B）可诱导的CYP的示例布局包括用于介质空白与探针孵育，并与探针和抑制剂孵育的一系列孔，代谢探针仅控制，诱导细胞（ 例如 ，用TCDD）。 <img alt="表2" src="/files/ftp_upload/55502/55502table2.jpg" /> 表2: 用于校准曲线以及相应的检测限和定量限合成标准稀释 。使用稀释范围可基于质量分析平台和灵敏度上。在这里，我们使用连接到UPLC混合三重四极/线性离子阱质谱仪。 <a href="http://ecsource.jove.com/files/ftp_upload/55502/55502table2large.jpg" target="_blank">请点击此处查看这个更大的版本</a>数字。 <img alt="表3" src="/files/ftp_upload/55502/55502table3.jpg" /> 表3:发光的探针，诱导剂和缓蚀剂的CYP1A1 / 1B1测定。 <a href="http://ecsource.jove.com/files/ftp_upload/55502/55502table3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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AB和EM是英美烟草公司的员工在这个稿子写的时间。在这个手稿中描述的工作是由英美烟草资助，并为这个视频文章的出版费用由英美烟草支付。

目前，许多标准化毒理学测试系统使用工程菌，细胞系，或胚胎细胞是不能代表正常人体新陈代谢1。这可能导致化学或医学的潜在的毒性活动的不准确的预测。 在体外模型中 ，如三维细胞培养物和器官在一个芯片上的创新，在尝试更好地复制人体组织21，22，23的正常形态和代谢活性被开发。代谢能力是可以用来破译这些机型最适合用于毒理评估和的药物生物转化研究的一个标准。 我们在这个文件中提出的方案是用来测量使用活细胞的CYP酶的活性。它是柔性的并且可用于在二维或三维文化性不同的细胞系统ES并提供了使用一个发光的探针 - 或质谱为基础的方法的选项。这两种方法足够敏感以检测材料纳克量，只需要少量在多孔格式中生长的细胞的。它们也可以应用到球状体或复杂基质中生长的细胞。探针基底的选择显然是该协议的一个关键因素。所述测定的特异性依赖于探针具有选择性的CYP酶和保证的另一层可以通过使用选择性CYP抑制剂来获得。底物和本文列出的抑制剂是只是几个例子，但是其他人可以在文献中找到。 用新的细胞类型为负CYP活动成果工作时，酶在较低水平被表达可能是由于考虑多种孵育时间是很重要的。添加可使用的作为阳性对照的细胞类型的建议，以确保一阴性结果未链接到一个技术问题，并与任何以帮助解决问题。原发性肝细胞代谢能力和可以被用作控制，用于在悬浮例如原代肝细胞是非常容易使用，但它们的生存能力是在时间上有限的。肝细胞系是它们相对容易，因为在这个方案中所述使用可能的替代方案，但在代谢活性6,21方面一些比另一些更好。 由于该测定是无损的，除非总蛋白进行定量，能够追随随时间的CYP活性在纵向研究20。这一点很重要，因为某些细胞会具有可变CYP活动在在培养时间。阴性结果可能与缺乏汇合，进步在体外分化或去分化有关。在这种情况下，该方法为半quantitati已经由于数据不被蛋白在各细胞培养物的总量归一化。这不总是可能的或推荐的测量CYP活性暴露于探针，抑制剂或诱导可对组织具有毒性作用如对于TCDD的情况之后重新使用组织。 细胞级分如微粒也可用于表征一个给定的细胞类型的CYP活性。微粒制剂，然而，需要大量的细胞材料，添加适当的辅因子和缓冲，以确保酶保持活性。使用活细胞消除了棘手的微粒准备步骤和工作哪个缓冲和辅助因子效果最好。 作为一般的建议，这是进一步表征的CYP的表达谱中的细胞培养物，以验证它相匹配的酶活性的最佳实践。这种额外的验证级别建议鉴于代谢探头不ENTI依靠特定于单个CYP，它给增加的置信度所测量的代谢活性被相应的酶表达匹配。由于的CYP为膜蛋白它们相对难以用于蛋白质印迹准备，因此定量RT-PCR已剖析这些酶20的优选方法。 要注意，该协议描述了测定CYP酶的活性，仅表示代谢酶中的一种家族，虽然是重要的一项所述的方法是重要的。质谱方法的灵活性允许为探针底物的其他代谢转化采集方法的发展。然而，这些已被开发一次一个，并且当前有较少的已知的特定的探针和用于其他代谢酶家族选择性抑制剂。这种差距应该给允许在体外细胞SYSTE的代谢能力进行全面评估女士。

异生素代谢是通过该化学品外国到主体通过加入亲水性基团和缀合物的修饰，以促进其排泄1的过程。典型地，外源物的代谢是与I由具有添加一个或多个羟基1,2的氧化的主要相的两步骤的过程。在第二阶段中，使用羟基作为受体为亲水性共轭，如葡糖苷酸或硫酸酯部分含1，2。如果一个受体基团是已经存在于分子中，结合步骤可独立阶段I代谢的发生。各反应通过一组特定的酶， 例如 ，的CYP（细胞色素P450的），即催化羟基化（ 图1）和脱烷基化化学基片3的执行。 Conjugation由磺基转移酶催化的，UDP-葡糖醛酸（ 图1），谷胱甘肽-S-转移酶和N-乙酰转移酶4。每个组织和器官将有一个特定的代谢酶的表达谱，用表达最那些蛋白质的肝脏。 <img alt="图1" src="/files/ftp_upload/55502/55502fig1.jpg" /> 图1:阶段I和阶段II代谢香豆素的实施例。 CYP2A6 / CYP2A13催化香豆素7-羟化。 7-羟基香豆素是由第II相酶转移酶缀合于葡萄糖醛酸部分，与UGT1A6和UGT1A9显示出最高的活性。 了解外源性物质的代谢是在药物安全性评价及化学品风险评估的原因有两个关键:反应动力学将确定药物或化学多久会留在体内的活性或非活性形式B安伏排泄;和母体化合物可通过代谢酶被修饰以一个更具反应性的，不稳定的和有毒的物质。这样的反应也被称为"生物活化"主要是由相位我CYP酶还基于通过相位共轭II 5罕见的场合驱动。 在此基础上， 在体外模型的精确预测与药物或化学相关的风险的能力是高度依赖于电池系统的代谢能力。从患病组织或从正常细胞的转化的细胞系常常失去部分即使不是所有的代谢代表原籍6的组织的酶轮廓。正常代谢酶轮廓的维护出现在原代细胞培养更好（至少在短期培养物），并且如果所述组织是在基质允许它保持其三维结构1培养的进一步提高。因此，焦炭体外细胞系统的代谢能力的acterization是指导有关决定哪些细胞模型适合于进行化学安全性评估的一个重要预备步骤。 在本文中，我们提出了一个协议与它们与肝细胞系7和3D主肺细胞培养物8应用实例来分析在体外的活性和期CYP酶的表达。 CYP特异性底物，其代谢产物和抑制剂控制是与质量spectrometry-和发光的基础的定量方法沿着所述。由于一些的CYP是诱导和其他人组成，例子也将给予这两个场景。

<table><tbody><tr><td>&lt;strong&gt;Reagent &amp;amp; Cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2 &amp;mu;m syringe filter</td><td>Whatman</td><td>UN203NPEORG</td><td></td></tr><tr><td>24 well plate</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>4-methylumbelliferone</td><td>Sigma Aldrich</td><td>M1381</td><td></td></tr><tr><td>5-phenyl pentyne</td><td>Sigma Aldrich</td><td>CD5001437</td><td></td></tr><tr><td>6-hydroxybupropion</td><td>Sigma Aldrich</td><td>H3167</td><td></td></tr><tr><td>6-hydroxychlorzoxazone</td><td>Sigma Aldrich</td><td>UC148</td><td></td></tr><tr><td>7-ethoxycoumarin</td><td>Sigma Aldrich</td><td>195642</td><td></td></tr><tr><td>7-hydroxycoumarin</td><td>Sigma Aldrich</td><td>H24003</td><td></td></tr><tr><td>8-methoxypsoralen</td><td>Sigma Aldrich</td><td>M3501</td><td></td></tr><tr><td>acetic acid</td><td>Sigma Aldrich</td><td>695092</td><td></td></tr><tr><td>acetonitrile</td><td>Fisher</td><td>A/0626/17</td><td></td></tr><tr><td>&amp;alpha;-naphthoflavone</td><td>Sigma Aldrich</td><td>N5757</td><td></td></tr><tr><td>BCA kit</td><td>Thermo Scientific</td><td>23227</td><td></td></tr><tr><td>b-glucuronidase/arylsulfatase</td><td>Sigma Aldrich</td><td>G0876</td><td></td></tr><tr><td>Bupropion</td><td>Sigma Aldrich</td><td>B102</td><td></td></tr><tr><td>Carbamazepine</td><td>Sigma Aldrich</td><td>C4024</td><td></td></tr><tr><td>chlorzoxasone</td><td>Sigma Aldrich</td><td>C4397</td><td></td></tr><tr><td>collagen</td><td>Cell Systems</td><td>5005-B</td><td></td></tr><tr><td>coumarin</td><td>Sigma Aldrich</td><td>C4261</td><td></td></tr><tr><td>disulfiram</td><td>Sigma Aldrich</td><td>86720</td><td></td></tr><tr><td>fluvoxamine</td><td>Sigma Aldrich</td><td>F2802</td><td></td></tr><tr><td>Glutamax (Glutamine supplement)</td><td>Fisher</td><td>35050061</td><td></td></tr><tr><td>HepaRG metabolism supplement</td><td>Merck</td><td>MMAD621</td><td></td></tr><tr><td>HepaRG thaw media supplement</td><td>Merck</td><td>MMADD671</td><td></td></tr><tr><td>HepaRG</td><td>Merck</td><td>MMHPR116</td><td></td></tr><tr><td>Luciferin-CEE</td><td>Promega</td><td>V8751</td><td></td></tr><tr><td>methanol</td><td>Fisher</td><td>M/4056/17</td><td></td></tr><tr><td>MucilAir airway cells</td><td>Epithelix</td><td>EP01</td><td></td></tr><tr><td>MucilAir airway cells maintenance media</td><td>Epithelix</td><td>EP04MM</td><td></td></tr><tr><td>Phenomenex Kinetex 2.6 &amp;mu;m, PFP 100A</td><td>Phenomenex</td><td>00B-4477-AN</td><td></td></tr><tr><td>TCDD</td><td>Sigma Aldrich</td><td>48599</td><td></td></tr><tr><td>thioTEPA</td><td>Sigma Aldrich</td><td>T6069</td><td></td></tr><tr><td>Waters Acquity UPLC HSS T3 1.8 &amp;micro;m 2.1 x 50 mm</td><td>Waters</td><td>86003538</td><td></td></tr><tr><td>Williams&amp;rsquo; E media</td><td>Fisher</td><td>17704-024</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>UPLC</td><td>Waters</td><td>Acquity</td><td></td></tr><tr><td>QTRAP MS</td><td>Sciex&amp;nbsp;</td><td>ABI Sciex 4000</td><td></td></tr><tr><td>QTRAP MS software</td><td>Sciex&amp;nbsp;</td><td>Analyst 1.4.2</td><td></td></tr><tr><td>luminometer</td><td>Molecular Devices</td><td>SpectraMax M3</td><td></td></tr><tr><td>spectrophotometer</td><td>Molecular Devices</td><td>SpectraMax M3</td><td></td></tr><tr><td>cell counter</td><td>Beckman Coulter</td><td>Vi-Cell XR</td><td></td></tr><tr><td>rotary evaporator</td><td>Eppendorf</td><td>Eppendorf-5301</td><td></td></tr></tbody></table>

mass spectrometry and luminogenic-based approaches to characterize phase i metabolic competency of in vitro cell cultures

异生代谢酶，通过添加提高溶解度和促进排泄的官能团发挥药物和有毒物质的生物转化的关键功能。在某些情况下这些结构的改变导致的新的有毒产物的生成。为了减少动物试验，化学危险，可以通过代谢感受态细胞进行评估。代谢酶的表达，然而，这不是随时间稳定在许多体外原代培养系统和经常是在细胞系中的部分或不存在。因此，药品，添加剂，并在体外环境污染物代谢的研究应当理想地在细胞系统中，其中代谢活性进行了表征进行。我们在这里介绍的方法使用UPLC质谱定量的化学探针及其代谢产物来衡量一类代谢酶的2D细胞系和原代3D文化活动（人类一期）和辉度。该方法可被实现，以测试在细胞系和来自各种组织来源的原代细胞的代谢活性。

两对的CYP的（CYP1A1 / 1B1和CYP2A6 / 2A13）在气道细胞培养物来自三个不同供体（气道M360，M370，M417）和肝细胞系（喉癌线）测量的代谢活性的例子示于图6中 20。 CYP活性通过辉度测定 图6A示出了由吕克-CEE（荧光素6&#39;-氯乙基醚）在气道细胞（3个供体）和肝细胞具有和不TCDD诱导CYP1A1 / 1B1 O型脱烷基化活性产生的相对发光。 CYP1A1 / 1B1是高度可诱导的酶，并且通常不是在大多数组织中组成型表达水平。如在图6A中可以看出，当细胞未被诱导没有检测到发光的信号。当两种细胞类型预孵育TCDD，强力CYP1A1 / 1B1诱导剂，标记与对照相比（ 图6A）被检测到ED发光的信号。这表明，吕克 - 中欧和东欧探头已经代谢导致其随后被量化的荧光素部分的释放。整体从TCDD处理的肝细胞系的活性比对气道细胞下，即使总蛋白归一化之后。这是不寻常的，因为细胞系往往不太代谢比主细胞中有活性。 α萘黄酮的添加具有其中进一步证实吕克-CEE代谢的CYP依赖性的所有细胞类型上CYP1A1 / 1B1活性的明确的抑制作用。 使用UPLC-MS CYP450代谢物的定量 图6B示出培育后CYP2A6 / 2A13活性的气道细胞从3个供体（气道M343，M345，M347）和肝细胞病毒（Hep线）定量在存在香豆素和不存在的INHIBITOR 8-甲氧基补骨脂。数据被归一化的总蛋白。在0分钟香豆素孵化对照，无7-羟基香豆素被检测到（ 图6B）。 CYP2A6 / 2A13在某些组织中组成型表达和在所测试的不同细胞类型中检测与形成香豆素7-羟基香豆素的后因此孵育16小时，即使没有诱导。当8-甲氧基补骨脂，一个CYP2A抑制剂加入（ 图6B），7-羟基香豆素的形成显着降低。 应当注意的是，在CYP抑制剂的存在，这是正常的还是检测一些残余活性抑制是很少完整并且由于其它的CYP也可能能够识别该基板但远不如有效地处理它。它也可以被观察到，使用的原代细胞时，因为它是在气道细胞CYP2A6可见记录的代谢活性可以是供体之间不同（ 图6B）。这是预期的CYP活性很容易给每个捐赠者的基因型和多态性。 <img alt="图6" src="/files/ftp_upload/55502/55502fig6.jpg" /> 图6:活性测定结果为CYP1A1 / 1B1活性（A），并在肝细胞系并在三气道细胞捐献者（M360，M370，M417）CYP2A6 / 2A13活性（B）。 （A）吕克-CEE脱烷基化活性显示具有和不具有诱导CYP1A1 / 1B1的由TCDD。 α萘黄酮用作CYP1A1 / 1B1抑制剂。活性表示为通过温育时间（分钟）（分钟）归一化相对发光单位（RLU）和总蛋白（毫克）。因为它们是组成型表达（B）CYP2A6 / 2A13不需要感应。以pmol /分钟/ mg蛋白香豆素7-羟基化活性被示出在有和没有抑制剂8-methoxypsor不同时间点阿伦。所有结果表示为与平均值的标准差三个独立测量的平均值。这个数字是从巴克斯特20修改。 参考表1:UPLC和质谱仪设置。 <a href="http://ecsource.jove.com/files/ftp_upload/55502/Supplemental_Table_1.xlsx" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures at JoVE.com

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

体外系统的代谢能力是药物和毒物的生物转化和处置的一个关键要求。在这个协议中，我们描述的参考代谢探针的应用，以评估相我在细胞培养物代谢。

质谱法和基于发光的-途径表征阶段我代谢能力的<em&gt;体外</em&gt;细胞培养

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

mass-spectrometry-luminogenic-based-approaches-to-characterize-phase

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

9.7K 次观看.  British American Tobacco.  体外系统的代谢能力是药物和毒物的生物转化和处置的一个关键要求。在这个协议中，我们描述的参考代谢探针的应用，以评估相我在细胞培养物代谢。 

视频: Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

在无菌无鼠标进定植评估肝代谢的变化 1 H核磁共振光谱

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

科研

免疫与感染

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

毛细管电泳质谱法提取培养细胞中的水代谢物进行代谢组学分析

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

癌症研究

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

FIBS功能的无创性代谢轮廓

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

生物工程

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

生物化学

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

集成细胞操作平台与单悬架细胞中药物和代谢物质谱分析的单探针相结合

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

化学

稳定同位素追踪和LC-MS分析以测量DHODH和SDH活性

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

测量哺乳动物线粒体功能的氧非依赖性测定

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

基于莱萨特的无细胞系统中的代谢物分析液色谱耦合折射指数或质谱检测

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

质谱法和基于发光的-途径表征阶段我代谢能力的

摘要

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此视频中的章节

在无菌无鼠标进定植评估肝代谢的变化¹ H核磁共振光谱

毛细管电泳质谱法提取培养细胞中的水代谢物进行代谢组学分析

FIBS功能的无创性代谢轮廓

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

集成细胞操作平台与单悬架细胞中药物和代谢物质谱分析的单探针相结合

测量哺乳动物线粒体功能的氧非依赖性测定

测量哺乳动物线粒体功能的氧非依赖性测定

测量哺乳动物线粒体功能的氧非依赖性测定

基于莱萨特的无细胞系统中的代谢物分析液色谱耦合折射指数或质谱检测

果蝇幼虫和成年脑的代谢分析