这份手稿中产生的人物是用 BioRender.com 创作的。我们感谢 Amy Walker 对本文提供反馈。J.B.S.得到了伍斯特生物医学研究基金会资助。

稳定同位素追踪和LC-MS分析以测量DHODH和SDH活性

<ol>
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作者没有利益冲突需要报告。

随着新兴研究表明哺乳动物线粒体可以在不消耗分子氧的情况下发挥作用，研究人员在OCR测量之外采用正交测定来准确量化线粒体功能至关重要。在这里，我们编制了一系列可用于通过测量线粒体NAD + / NADH平衡，自适应末端电子受体的利用，ATP的产生，从 头 嘧啶生物合成和线粒体衍生的ROS来直接评估复合物I，复合物II，复合物V和DHODH的活性。值得注意的是，这些测定比OCR测量更直接地测量线粒体功能。此外，这些测定为研究人员提供了在缺氧期间量化线粒体功能的易处理方法，由于富马酸盐被用作首选的末端电子受体，因此 OCR 测量在很大程度上无关紧要。最后，这里描述的基于增殖的方法比经典的呼吸测量实验更具成本效益，从而提供了一种广泛可访问的方法来研究哺乳动物系统中的线粒体功能。
利用这些检测来测量培养细胞中的线粒体功能时，有一些关键的考虑因素。关于增殖测定，重要的是调整接种的细胞数量以增加每个细胞系的倍增率。应将细胞接种至至少10%的汇合度，并有足够的空间允许三到四倍，以便可以量化增殖差异。每种测定的另一个考虑因素是用作每个ETC复合物活性对照的小分子的浓度。由于不同的细胞系可能对这些抑制剂表现出不同的敏感性，因此测试这些小分子的剂量以确定最佳浓度至关重要。
体外研究线粒体功能的测定（包括OCR测量和此处描述的所有测定）的普遍局限性是培养基的代谢组成。标准细胞培养基倾向于使系统偏向于表面高水平的线粒体功能。例如，超生理谷氨酰胺水平增加了其TCA循环25的无激素，这促进了线粒体NADH的合成，从而增加了氧化磷酸化。同样，哺乳动物组织中的氧分压范围在3mmHg和100mmHg（约0.1%-13%O2）之间，但在体外是大气压（140mmHg，约21%）26，27。这种过量的O2使线粒体呼吸能力和超氧化物产生最大化28。最近，人们努力设计更生理的培养基29，30。值得注意的是，在人血浆样培养基中培养细胞会降低某些癌细胞系的线粒体呼吸30，T细胞31中的线粒体ROS以及线粒体对癌症治疗的适应32。因此，注意所用培养基的组成并了解其如何影响线粒体功能至关重要。
解释线粒体功能的另一个重要和普遍的限制是线粒体数量差异的可能性。因此，通过定量mtDNA33、用膜电位不敏感染料34测量线粒体质量或线粒体标志物的蛋白质印迹来测量线粒体含量至关重要。这是一个关键的控制，因此线粒体数量的减少不会被误认为线粒体功能的下降。
还有一些适用于此处描述的测定的特定限制和故障排除。首先，鉴于分化的细胞不会增殖，在这种情况下，基于增殖的测定对于评估线粒体功能没有用。用于测量DHODH活性的 13C4-天冬氨酸示踪方案的一个关键限制是细胞中的天冬氨酸摄取效率极低35。为了克服这一潜在的限制，研究人员可以过表达天冬氨酸转运蛋白SLC1A3，以促进 13C4-天冬氨酸摄取35。
使用13C5-谷氨酰胺示踪测量SDH活性的方案的局限性在于，该测定要求细胞利用还原羧化途径来富集M + 3同位素异构体以测量反向活性。由于ATP柠檬酸裂解酶表达低36，HIF稳定性不足37或α-KG：柠檬酸盐比值太低38，一些细胞系无法进行还原羧化通量。为了克服这一限制，可以利用13C4-天冬氨酸示踪来测量SDH正向和反向活动7。在该测定中，SDH正向活性可以通过富马酸盐M + 2：琥珀酸M + 2的比率和琥珀酸盐M + 4：富马酸盐M + 4的反向反应来测量。值得注意的是，这种示踪绕过了还原羧化途径中的大多数酶。
使用DCPIP还原作为读数的复合物I活性测定的局限性是线粒体在结构上不是完整的。冻融线粒体以使NADH吸收用于测定的过程肯定会损害线粒体膜的结构完整性39。该测定应与诸如复合物I增殖测定之类的测定同时进行，以确保观察到的复合物I活性的变化对于完整细胞也是真实的。
在未来的研究中，其中一些技术可以适用于使用模型生物（如小鼠和秀丽隐杆线虫）测量体内线粒体功能。目前用于测量体内线粒体功能的方法集中在生物体水平的OCR，特别是使用小鼠模型时的呼吸交换率。这种方法的一个明显局限性是，氧气除了在线粒体ETC中作为无处不在的末端电子受体的作用外，还具有许多生化和信号功能。例如，氧气被双加氧酶家族中酶的催化活性“消耗”。虽然这些酶有助于细胞耗氧率，但它们不参与、调节或反映线粒体功能。体外经典呼吸测量实验通常控制“非线粒体OCR”，而有机呼吸交换比（RER）实验无法控制这一点，限制了RER作为体内线粒体功能的指标的解释。然而，调整方案以通过13C4-天冬氨酸示踪测量DHODH活性，通过13C5-谷氨酰胺示踪测量复合物II活性，从组织中纯化的线粒体上的复合物I活性，以及使用LC-MS友好化合物（如MitoB）测量线粒体ROS以测量体内线粒体功能.这些询问线粒体功能的直接测定，结合经典的呼吸测量实验，为研究人员提供了对哺乳动物细胞和组织中线粒体功能的更全面和准确的评估。

线粒体功能是细胞健康的关键指标，因为它维持哺乳动物细胞中关键的生物合成、生物能量和信号功能1。绝大多数线粒体功能需要电子流过电子传递链（ETC），ETC中电子流的中断会导致严重的线粒体疾病2。ETC由嵌入线粒体内膜中的一系列还原和氧化（氧化还原）反应组成，这些电子转移反应释放的自由能可用于支持ATP合成，生理过程（例如产热），生物合成途径（例如从头嘧啶生物合成）以及辅助因子（例如NADH）的氧化还原状态的平衡。ETC复合物I和III产生活性氧（ROS）3，4，5，进而调节信号传导关键途径，如HIF，PI3K，NRF2，NFκB和MAPK6。因此，ETC中的电子流指标通常用作哺乳动物细胞中线粒体功能的代理。
呼吸测量实验经常用于测量哺乳动物细胞中的线粒体功能。由于O2是哺乳动物ETC最普遍的末端电子受体，因此其还原被用作线粒体功能的代表。然而，新出现的证据表明，哺乳动物线粒体可以使用富马酸盐作为电子受体来维持依赖于ETC的线粒体功能，包括从头嘧啶生物合成7，NADH氧化7和硫化氢的解毒8。因此，在某些情况下，特别是在缺氧环境中，O2消耗率（OCR）的测量不能提供线粒体功能的精确或准确的指示。
在这里，我们概述了一系列可用于独立于OCR测量线粒体功能的测定。我们提供直接测量复合物 I 介导的 NADH 氧化、二氢乳清酸脱氢酶介导的 从头 嘧啶生物合成、复合物 V 依赖性 ATP 合成、琥珀酸脱氢酶 （SDH） 复合物的净方向性和线粒体衍生的 ROS 的检测方法。这些测定旨在对培养的哺乳动物细胞进行，尽管许多测定可以适应 研究体内 线粒体功能。值得注意的是，该协议中描述的测定是比OCR更直接的线粒体功能测量。此外，它们能够测量缺氧时的线粒体功能，在这种情况下，OCR 不是指示性测量。总之，这些测定与经典的呼吸测量实验相结合，将为研究人员提供对哺乳动物细胞线粒体功能的更全面的评估。

<table><tbody><tr><td>&amp;nbsp;1.5 mL tube</td><td>Cell Treat</td><td>667443</td><td></td></tr><tr><td>2.0 mL tube</td><td>Cell Treat</td><td>229446</td><td></td></tr><tr><td>6-well plate</td><td>Cell Treat</td><td>229106</td><td></td></tr><tr><td>12-well plate</td><td>Cell Treat</td><td>229112</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;4&lt;/sub&gt;-aspartate</td><td>Sigma-Aldrich</td><td>604852</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;5&lt;/sub&gt;-Glutamine</td><td>Cambridge Isotope Laboratories</td><td>285978-14-5</td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>Cell Treat</td><td>667411</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Cell Treat</td><td>667421</td><td></td></tr><tr><td>150 mm tissue culture dish</td><td>Cell Treat</td><td>229651</td><td></td></tr><tr><td>1x Phosphate-buffered saline</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>2,6-dichlorophenolindophenol</td><td>Honeywell</td><td>33125</td><td></td></tr><tr><td>Ammonium Carbonate</td><td>Sigma-Aldrich</td><td>37999</td><td></td></tr><tr><td>Antimycin</td><td>Sigma-Aldrich</td><td>A8674</td><td></td></tr><tr><td>Ascentis Express C18</td><td>Sigma-Aldrich</td><td>53825-U</td><td></td></tr><tr><td>Bottle top filter 500 mL, 0.22 &amp;micro;m, PES 9 9 mm membrane diameter</td><td>Cell Treat</td><td>229717</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A3294</td><td></td></tr><tr><td>Brequinar</td><td>Sigma-Aldrich</td><td>SML0113</td><td></td></tr><tr><td>Cell Lifter, Double End Flat and Narrow Blade</td><td>Cell Treat</td><td>229305</td><td></td></tr><tr><td>CentriVap -105 Cold Trap</td><td>Labconco</td><td>7385020</td><td></td></tr><tr><td>Complete Protease Inhibitor Tablets</td><td>Sigma-Aldrich&amp;nbsp;</td><td>4693116001</td><td></td></tr><tr><td>Coulter Counter Cups</td><td>Fisher Scientific</td><td>07-000-694</td><td></td></tr><tr><td>Decylubiquinone</td><td>Sigma-Aldrich</td><td>D7911</td><td></td></tr><tr><td>DMSO</td><td>Invitrogen</td><td>D12345</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Eppendorf Centrifuge 5425R</td><td>Eppendorf</td><td>2231000908</td><td></td></tr><tr><td>Eppendorf Centrifuge 5910 Ri</td><td>Eppendorf</td><td>5943000343</td><td></td></tr><tr><td>Galactose</td><td>Sigma-Aldrich</td><td>G5388</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Glucose-free DMEM</td><td>Gibco</td><td>11966025</td><td></td></tr><tr><td>Glutamine-free DMEM</td><td>Thermo Fisher</td><td>11960044</td><td></td></tr><tr><td>Heat-Inactivated Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F4135</td><td></td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>HPLC-grade 35% Ammonium hydroxide</td><td>Thermo Scientific</td><td>460801000</td><td></td></tr><tr><td>HPLC-grade Acetonitrile</td><td>Sigma-Aldrich</td><td>900667</td><td></td></tr><tr><td>HPLC-grade Chloroform</td><td>Sigma-Aldrich</td><td>366927</td><td></td></tr><tr><td>HPLC-grade formic acid</td><td>Thermo Scientific</td><td>28905</td><td></td></tr><tr><td>HPLC-grade Isopropanol</td><td>Sigma-Aldrich</td><td>563935</td><td></td></tr><tr><td>HPLC-grade MeOH</td><td>Sigma-Aldrich</td><td>900688</td><td></td></tr><tr><td>HPLC-grade Water</td><td>Sigma-Aldrich</td><td>270733</td><td></td></tr><tr><td>Human Osteosarcome Cell Line 143B</td><td>ATCC</td><td>CRL-8303</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Sigma-Aldrich</td><td>320331-500ML</td><td></td></tr><tr><td>Isotone buffer</td><td>Beckman Coulter</td><td>8546719</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P2222</td><td></td></tr><tr><td>Mannitol</td><td>Sigma-Aldrich</td><td>M4125</td><td></td></tr><tr><td>MitoSox Red</td><td>Invitrogen</td><td>M36008</td><td></td></tr><tr><td>N-acetyl-L-cysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>Oligomycin</td><td>Sigma-Aldrich</td><td>75351-5MG</td><td></td></tr><tr><td>Pencillin Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Potter-Elvehjem Tissue Grinder, Size 21</td><td>Kimble</td><td>885502-0021</td><td></td></tr><tr><td>Pyruvate</td><td>Sigma-Aldrich</td><td>P5280</td><td></td></tr><tr><td>Pyruvate-free DMEM media</td><td>Gibco</td><td>11965175</td><td></td></tr><tr><td>Q Exactive Plus Mass Spectrometer</td><td>Thermo Scientific</td><td>726030</td><td></td></tr><tr><td>ReCO&lt;sub&gt;2&lt;/sub&gt;ver Incubator</td><td>Baker</td><td></td><td></td></tr><tr><td>Refrigerated Centrivap Benchtop Vacuum Concentrator</td><td>Labconco</td><td>7310020</td><td></td></tr><tr><td>RIPA Buffer</td><td>Millipore Sigma</td><td>20188</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>SeQuant ZIC-pHILIC 5&amp;mu;m 150 x 2.1 mm analytical column</td><td>Sigma-Aldrich</td><td>1.50460.0001</td><td></td></tr><tr><td>SeQuant ZIC-pHILIC guard kit</td><td>Millipore Sigma</td><td>1.50438.0001</td><td></td></tr><tr><td>Sodium Hydroxide, Pellets</td><td>Millipore Sigma</td><td>567530-250GM</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>SW, TRACEFINDER 5.1 SP3</td><td>Thermo Scientific</td><td>OPTON-31001</td><td></td></tr><tr><td>Tert-butyl hydroperoxide solution</td><td>Sigma-Aldrich</td><td>458139</td><td></td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>93352</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Gibco</td><td>25-200-114</td><td></td></tr><tr><td>Uridine</td><td>Sigma-Aldrich</td><td>U3003</td><td></td></tr><tr><td>VANQUISH HORIZON / FLEX HPLC</td><td>Thermo Scientific</td><td>VF-S01-A-02</td><td></td></tr><tr><td>Z2 Coulter Particle count and size analyzer</td><td>Beckman Coulter</td><td>BZ10131270</td><td></td></tr></tbody></table>

oxygen-independent assays to measure mitochondrial function in mammals

线粒体电子传递链（ETC）中的电子流动支持哺乳动物细胞中的多方面生物合成，生物能量和信号传导功能。由于氧（O 2）是哺乳动物ETC最普遍的末端电子受体，因此O2消耗率经常被用作线粒体功能的代表。然而，新兴研究表明，该参数并不总是指示线粒体功能，因为富马酸盐可以用作替代电子受体以维持缺氧时的线粒体功能。本文编制了一系列协议，允许研究人员独立于O2消耗率测量线粒体功能。这些测定在研究缺氧环境中的线粒体功能时特别有用。具体来说，我们描述了测量线粒体ATP产生，从头嘧啶生物合成，复合物I氧化NADH和超氧化物产生的方法。结合经典的呼吸测量实验，这些正交和经济的测定将为研究人员提供更全面的线粒体功能评估他们感兴趣的系统。

DHODH，复合物I和复合物V的活性都可以使用增殖测定进行评估。从培养基中剥夺尿苷后，细胞变得更加依赖于嘧啶生物合成的 从头 途径。因此，当细胞在无尿苷培养基中被激发增殖时，它们比在含有尿苷的培养基中培养的细胞对brequinar抑制DHODH活性更敏感（图6A）。类似地，从培养基中剥夺丙酮酸使细胞更依赖于复合物I活性进行增殖。因此，当细胞在无丙酮酸培养基中被激发增殖时，它们比在含丙酮的培养基中培养的细胞对鱼藤酮对复合物I活性的抑制更敏感（图6B）。复合V活性可以通过挑战细胞在含有半乳糖而不是葡萄糖的培养基中增殖来评估。由于半乳糖在糖酵解中产生净零ATP，因此在这种燃料中生长的细胞更依赖于通过复合V活性合成线粒体ATP。因此，在含半乳糖培养基中增殖的细胞比在含葡萄糖培养基中增殖的细胞对寡霉素的复合物V抑制更敏感（图6C）。
SDH活性可以使用13C5-谷氨酰胺示踪和监测其掺入富马酸盐和琥珀酸盐同位素异构体来测量。在载体处理条件下，SDH复合物有利于前向活性，将13C 4-琥珀酸掺入13 C4-富马酸盐高于将13 C 3-富马酸盐掺入13 C3-琥珀酸盐（图7）。在抗霉素处理的条件下，SDH复合物有利于反向活性，并且将13C 3-富马酸盐掺入13 C3-琥珀酸盐大于将13 C 4-琥珀酸掺入13 C4-富马酸盐（图7）。
线粒体内的超氧化物产生可以使用荧光报告基因MitoSox进行测量，其在与超氧化物反应时产生2-羟基-丝分裂乙铓。在这项研究中，在叔丁基过氧化氢存在下用MitoSox处理的细胞具有更高水平的2-羟基-丝分裂乙锭，其方式通过添加NAC（一种淬灭细胞ROS的抗氧化剂）来抑制（图8）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65184/65184fig01.jpg"> 图 1：复杂 V 增殖测定的机理基础。 通过糖酵解氧化葡萄糖和半乳糖。葡萄糖从糖酵解中产生净两个ATP，而半乳糖产生净零ATP，因为UDP-半乳糖需要UTP合成。因此，在半乳糖中生长的细胞由于缺乏糖酵解产生的ATP，更依赖于线粒体ATP的合成。缩写：GALK = 半乳糖激酶;GALT = 半乳糖-1-磷酸尿苷基转移酶;PGM1 = 磷酸葡萄糖变位酶 1;GPI = 葡萄糖-6-磷酸异构酶，UGP = UDP-葡萄糖焦磷酸化酶;GALE = UDP-半乳糖-4-差向异构酶;NDK = 核苷酸二磷酸激酶;UMPK = 尿苷单磷酸激酶;HK = 己糖激酶;PFK = 磷酸果糖激酶;醛缩酶=醛缩酶;TPI = 磷酸三糖异构酶;GAPDH = 甘油醛-3-磷酸脱氢酶;PGK =磷酸甘油酸激酶;PGM = 磷酸葡萄糖变位酶;ENO = 烯醇化酶;PK = 丙酮酸激酶。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65184/65184fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65184/65184fig02.jpg"> 图2：复合物I增殖测定的机理基础。 在抑制复合物I活性时改变的代谢途径示意图。在高丙酮酸培养基中，通过LDH介导的NADH氧化绕过复合物I抑制。在低丙酮酸培养基中，这种适应不太可行，使细胞更依赖复合物I活性来再氧化NADH。缩写：LDH = 乳酸脱氢酶;TCA循环=三羧酸循环。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65184/65184fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65184/65184fig03.jpg"> 图 3： 13C4-天冬氨酸示踪时的 DHODH 反应示意图。 Brequinar抑制二氢乳清酸氧化为乳清酸，从而阻止UMP的下游合成。 13C4-天冬氨酸通过DHODH活性掺入 13C3-UMP中。缩写：OMM = 线粒体外膜;IMM = 线粒体内膜;DHODH = 二氢乳清酸脱氢酶。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65184/65184fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65184/65184fig04.jpg"> 图 4：通过 13C5-谷氨酰胺示踪测量正向和反向复合物 II 活性。为了测量正向复合物II活性（左），监测13C5-谷氨酰胺掺入13 C4-琥珀酸和13C4-富马酸盐。为了测量反向复合物II活性（右），监测13C5-谷氨酰胺掺入13 C 3-琥珀酸和13C3-富马酸盐。缩写：SDH = 琥珀酸脱氢酶;CytC = 细胞色素 C.<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65184/65184fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65184/65184fig05.jpg"> 图5：MitoSox Red与超氧化物的反应。 MitoSox与线粒体超氧化物反应形成2-OH-MitoE2+。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65184/65184fig05large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65184/65184fig06.jpg"> 图 6：用于测量线粒体功能的 基于增殖的测定 （A） 用 5 uM brequinar（一种 DHODH 抑制剂）处理的 143B 骨肉瘤细胞在含有 ±100 ug/mL 尿苷的培养基中的增殖。数据是平均± SEM;N = 3 每个条件。（B）用2uM鱼藤酮（一种复合物I抑制剂）处理的143B骨肉瘤细胞在含有±5mM丙酮酸的培养基中增殖。数据是平均± SEM;N = 3 每个条件。（C）用5 uM寡霉素（一种复合V抑制剂）处理的143B骨肉瘤细胞在以10 mM葡萄糖或10 mM半乳糖为唯一中心碳源的培养基中增殖。数据是平均± SEM;N = 3 每个条件。* 表示 p < 0.05，使用在 Graphpad Prism 中进行单因素方差分析检验。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65184/65184fig06large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65184/65184fig07.jpg"> 图 7： 13C5-谷氨酰胺示踪以测量复合物 II 活性。 DMSO和500nM抗霉素A处理的Caki1和DLD1细胞中的琥珀酸盐氧化和富马酸盐还原（SDH逆转）。数据代表平均± SEM;N = 3 每个条件。使用 GraphPad Prism 中的未配对 t 检验表示 p < 0.05。缩写：SDH = 琥珀酸氧化;SDH 逆转 = 富马酸盐减少。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65184/65184fig07large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65184/65184fig08v2.jpg"> 图 8：基于 LCMS 的 MitoSox 检测超氧化物。 在tBuOOH±NAC存在下，从用MitoSox处理的Caki1细胞中分离的2-OH-Mito E2+ 的提取离子色谱图30分钟。缩写：LCMS =液相色谱-质谱;NAC = N-乙酰半胱氨酸。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65184/65184fig08large.jpg" target="_blank">请点击此处查看此图的大图。</a>
表1：本协议中使用的试剂、缓冲液和培养基的组成。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65184/65184_Table%201%20Reaction%20Compositions_Revision%20(2).xlsx" target="_blank">请按此下载此表格。</a>

Watch this Scientific Journal Video about Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals at JoVE.com

作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

在这里，我们提出了一个测定汇编，以直接测量哺乳动物细胞中的线粒体功能，而与它们消耗分子氧的能力无关。

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 ...

我们的研究重点是线粒体如何感知和适应代谢压力源。一个关键的代谢应激源是缺氧，当细胞缺氧时就会发生缺氧。我们正在开发更好的检测方法来研究低氧环境中的线粒体功能。采用更高分辨率的质谱法研究细胞和组织代谢物水平。我们使用稳定同位素示踪研究来追踪特定代谢物的命运，并发现与健康和患病哺乳动物生理学相关的新代谢途径。研究小组发现，哺乳动物线粒体可以在低氧环境中维持关键功能。在机械上，他们使用富马酸盐作为电子传输链的替代电子权杖。该协议汇编了几种分析方法，研究人员可以使用这些测定来更好地评估哺乳动物细胞中的线粒体功能。重要的是，这些方案可用于研究缺氧时的线粒体功能，因为经典的呼吸测量实验是不够的。

oxygen-independent-assays-to-measure-mitochondrial-function-in-mammals

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

2.4K 次观看.  University of Massachusetts Chan Medical School. 我们的研究重点是线粒体如何感知和适应代谢压力源。一个关键的代谢应激源是缺氧，当细胞缺氧时就会发生缺氧。我们正在开发更好的检测方法来研究低氧环境中的线粒体功能。采用更高分辨率的质谱法研究细胞和组织代谢物水平。我们使用稳定同位素示踪研究来追踪特定代谢物的命运，并发现与健康和患病哺乳动物生理学相关的新代谢途径。研究小组发现，哺乳动?...

视频: 作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

科研

发育生物学

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

用最小组织量测定果蝇透纤维中线粒体耗氧量

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

生物化学

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule synthesis. Genetic and functional adaptations of mitochondria to the tumor environment drive proliferative and metastatic potential. The advent of DNA and RNA sequencing removed critical barriers to the evaluation of genetic mediators of tumorigenesis. However, to date, methodological approaches to evaluate tumor mitochondrial function remain elusive and require technical proficiency limiting the feasibility, ultimately diminishing diagnostic and prognostic value in both experimental and clinical settings. Here, we&#160;outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates using high-resolution respirometry. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways. Using this protocol, we demonstrate that mice bearing a luminal B mammary cancer exhibit defective nicotinamide adenine dinucleotide-linked respiration and reliance on succinate to generate adenosine triphosphate via OXPHOS.

We developed a practical protocol and analytical approach to evaluate mitochondrial oxidative phosphorylation and electron transfer capacity in fresh tumor homogenates. This protocol can be easily adapted to survey various mitochondrial functions that contribute to cancer initiation, progression, and treatment response.

分析测定切除实体瘤匀浆物的线粒体功能

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule ...

癌症研究

Mitochondrial Functional Assessment in Brush Lizard Liver Mitochondria

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of ...

<meta charset="utf8"></head><body>从啮齿动物中分离线粒体以进行高质量的生物能量研究

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

<meta charset="utf8"></head><body>作者聚焦:揭示线粒体磷酸钙在心力衰竭和生物能量学中的作用

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

从啮齿动物心脏组织中稳健地分离线粒体 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

生物学

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 ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

测量哺乳动物线粒体功能的氧非依赖性测定

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使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

用最小组织量测定果蝇透纤维中线粒体耗氧量

分析测定切除实体瘤匀浆物的线粒体功能

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

从啮齿动物心脏组织中稳健地分离线粒体

从啮齿动物心脏组织中稳健地分离线粒体

从啮齿动物心脏组织中稳健地分离线粒体

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

隔离和线粒体从培养细胞和小鼠组织功能分析

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体