开发移动式线粒体生理学实验室，用于在野外测量线粒体能量学
Summary
我们设计并建造了一个移动实验室，以测量在野外捕获的野生动物分离的线粒体的呼吸速率。在这里，我们描述了移动线粒体实验室的设计和装备以及相关的实验室协议。
Abstract
线粒体能量学是动物生物化学和生理学的中心主题，研究人员使用线粒体呼吸作为研究代谢能力的指标。为了获得线粒体呼吸的测量值，必须使用新鲜的生物样品，并且必须在约2小时内完成整个实验室程序。此外，需要多台专用设备来执行这些实验室检测。这给测量远离生理学实验室的野生动物组织中的线粒体呼吸带来了挑战，因为在野外收集活组织后无法保存很长时间。此外，长距离运输活体动物会引起压力，从而改变线粒体能量学。
这份手稿介绍了奥本大学 （AU） MitoMobile，这是一个移动的线粒体生理学实验室，可以带到现场并在现场用于测量从野生动物收集的组织中的线粒体代谢。介绍了移动实验室的基本特征以及测量分离线粒体呼吸速率的分步方法。此外，所提供的数据验证了装备移动线粒体生理学实验室和进行线粒体呼吸测量的成功。移动实验室的新颖之处在于能够开车到现场并对现场捕获的动物组织进行线粒体测量。
Introduction
迄今为止，旨在测量线粒体能量学的研究仅限于实验动物或在已建立的生理学实验室附近捕获的动物，这使得科学家无法对在迁徙、潜水和冬眠等活动中从动物身上收集的组织进行线粒体生物能量研究1,2,3,4,5,6 .虽然许多研究人员已经成功地测量了野生动物的基础和峰值代谢率以及每日能量消耗7,8，但研究人员测量线粒体性能的能力仍然有限（但见1,4,9）。这部分是由于需要用于分离线粒体的新鲜组织，以及在获得新鲜组织后约 2 小时内进行分离的实验室设施。一旦线粒体被分离，线粒体呼吸测量也应在~1小时内完成。
分离的线粒体呼吸速率通常通过测量连接到 Clark 电极的密封容器中的氧浓度来执行。这种方法背后的理论建立在基本观察的基础上，即氧气是氧化磷酸化过程中线粒体呼吸的最后一个电子受体。因此，当实验过程中氧浓度下降时，假设发生三磷酸腺苷（ATP）的产生10。消耗的氧气是产生的ATP的代名词。研究人员可以使用不同的底物创建特定的实验条件，并通过向腔室中添加预定量的 ADP 来启动二磷酸腺苷 （ADP） 刺激的呼吸（状态 3）。外源性ADP磷酸化为ATP后，耗氧率降低，达到状态4，可以测量。此外，添加特异性抑制剂可以获得有关漏呼吸和不偶联呼吸的信息10。状态 3 与状态 4 的比率决定了呼吸控制比率 （RCR），它是整体线粒体偶联的指标10,11。较低的 RCR 值表明整体线粒体功能障碍，而较高的 RCR 值表明线粒体偶联程度更大10。
如前所述，生物材料的收集、线粒体分离和呼吸速率的测量必须在获得组织后 2 小时内完成。为了在不将动物长途运输到已建立的实验室的情况下完成这项任务，建造了一个移动线粒体生理学实验室，将其带到可以收集这些数据的现场位置。2018 年的 Jayco Redhawk 休闲车被改装成移动分子生理学实验室，并命名为奥本大学 （AU） MitoMobile （图 1A）。之所以选择休闲车，是因为内置冰箱、冰柜、储水箱和管道、由 12 伏电池供电的电力、气体发生器、丙烷罐和自流平系统。此外，休闲车提供了在偏远地点过夜以收集数据的能力。车辆的前部没有改变，提供了驾驶和睡眠区 （图1B）。之前安装在车辆后部和炉灶的卧室设施（床、电视和橱柜）被拆除。
安装了定制的不锈钢搁板和由 80/20 铝框架支撑的定制石英台面，以代替卧室设施和炉灶 （图 1C）。实验室工作台为数据收集提供了足够的空间 （图1D）。每台设备（即冷冻离心机、线粒体呼吸室、酶标仪、计算机、均质机、秤、便携式超速冷冻机和其他一般实验室用品）的功耗都被考虑在内。为了支持离心机的大电压和电流需求，电气系统升级为航空级设备。车辆后部的外部隔间被改造成液氮储存舱，符合美国交通部的液氮储存和运输指南。该存储单元由不锈钢制成，并具有适当的通风功能，以防止任何膨胀的氮气泄漏到车辆的乘客舱中。
为了确认移动实验室可用于线粒体生物能量研究，分离了线粒体，并测量了野生家鼠（Mus musculus）后肢骨骼肌的线粒体呼吸速率。由于Mus musculus是一种模式生物，因此该物种的线粒体呼吸速率已确定为12,13,14。尽管先前的研究已经记录了通过差速离心进行线粒体分离15,16,17，但下文简要概述了移动线粒体生理学实验室方法中使用的方法。
Protocol
Representative Results
Discussion
移动线粒体生理学实验室使研究人员能够在远程现场收集组织后 2 小时内分离线粒体并测量线粒体呼吸速率。本文提出的结果表明，在AU MitoMobile中进行的线粒体呼吸测量与在大学研究实验室中进行的测量相当。具体来说，这里介绍的野生来源的Mus musculus的状态3，状态4和RCR的值与同一实验室和其他实验室先前发表的结果相当（表3）12,23</s…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢奥本大学塞缪尔·吉恩工程学院电气与计算机工程系的 Mark Nelms 和 John Tennant 帮助 AU MitoMobile 的结构和电气装备。此外，作者还感谢为AU MitoMobile提供的资金以及奥本大学跨学科研究总统奖（PAIR）资助的研究。
Materials
| 1.7 mL centrifuge tubes | VWR | 87003-294 | |
| 2.0 mL centrifuge tubes | VWR | 87003-298 | |
| 50 mL centrifuge tubes | VWR | 21009-681 | Nalgene Oak Ridge Centrifuge Tube |
| ADP | VWR | 97061-104 | |
| ATP | VWR | 700009-070 | |
| Bradford | VWR | 7065-020 | |
| Clear 96 well plate | VWR | 82050-760 | Greiner Bio-One |
| Dounce homogenizer | VWR | 22877-284 | Corning |
| EGTA | VWR | EM-4100 | |
| Filter paper | Included with Hansatech OxyGraph | ||
| Free-fatty acid BSA | VWR | 89423-672 | |
| Glucose | VWR | BDH8005-500G | |
| Glutamate | VWR | A12919 | |
| Hamilton Syringes | VWR | 60373-985 | Gaslight 1700 Series Syringes |
| Hansatech OxyGraph | Hansatech Instruments Ltd | No Catalog Number, but can be found under Products –> Electrode Control Units | |
| KH2PO4 | VWR | 97062-350 | |
| Malate | VWR | 97062-140 | |
| Mannitol | VWR | 97061-052 | |
| Membrane | Included with Hansatech OxyGraph | ||
| MgCl2 | VWR | 97063-152 | |
| MOPS | VWR | 80503-004 | |
| Policeman | VWR | 470104-462 | |
| Polytron | Thomas Scientific | 11090044 | |
| Potassium chloride (KCl) | VWR | 97061-566 | |
| Protease | VWR | 97062-366 | Trypsin is commonly used; however, other proteases can be used. |
| Pyruvic acid | VWR | 97061-448 | |
| Sodium Dithionite | VWR | AA33381-22 | |
| Succinate | VWR | 89230-086 | |
| Sucrose | VWR | BDH0308-500G | |
| Tris-Base | VWR | 97061-794 | |
| Tris-HCl | VWR | 97061-258 |
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