使用细胞外通量分析仪评估小鼠造血干细胞和原始祖细胞中的细胞生物能量学
Summary
这里介绍的方法总结了使用细胞外通量分析仪实时测量HSPCs的细胞外酸化速率（ECAR）和耗氧速率（OCR）评估非贴壁小鼠造血干细胞和原始祖细胞（HSPC）中细胞生物能量的优化方案。
Abstract
在稳态下，造血干细胞（HSC）在很大程度上保持静止，并且被认为主要依靠糖酵解来满足其能量需求。然而，在感染或失血等应激条件下，HSC增殖并迅速产生下游祖细胞，而下游祖细胞又进一步分化，最终产生成熟的血细胞。在这个转变和分化过程中，HSC从静止状态中退出，并迅速经历从糖酵解到氧化磷酸化（OxPHOS）的代谢转换。各种应激条件，如衰老，癌症，糖尿病和肥胖症，会对线粒体功能产生负面影响，从而改变造血过程中HSC和祖细胞的代谢重编程和分化。通过评估HSC和祖细胞在正常和胁迫条件下的糖酵解和线粒体功能，可以获得对其细胞外酸化率（ECAR）和耗氧率（OCR）的宝贵见解，这分别是细胞糖酵解和线粒体呼吸的指标。
在这里，提供了一个详细的方案，以使用细胞外通量分析仪测量小鼠骨髓来源的谱系阴性细胞群中的ECAR和OCR，其中包括造血干细胞和原始祖细胞（HSPC）。该协议描述了从小鼠骨髓中分离谱系阴性细胞的方法，解释了这些测定中使用的细胞接种密度和2-脱氧-D-葡萄糖（2-DG，抑制糖酵解的葡萄糖类似物）和各种OxPHOS靶向药物（寡霉素，FCCP，鱼藤酮和抗霉素A）浓度的优化，并描述了药物治疗策略。糖酵解通量的关键参数，如糖酵解、糖酵解容量和糖酵解储备，以及OxPHOS参数，如基础呼吸、最大呼吸、质子泄漏、ATP产生、备用呼吸能力和偶联效率，可以在这些测定中测量。该协议允许在非贴壁性HSPCs上进行ECAR和OCR测量，并且可以推广以优化任何类型悬浮细胞的分析条件。
Introduction
造血是由HSCs1形成具有高度专业化功能的各种类型的成熟血细胞的过程。HSC能够自我更新和分化成各种多能和谱系特异性祖细胞群。这些祖细胞最终产生淋巴样、髓系、红细胞和巨核细胞谱系的细胞。为了保持其自我更新能力，HSC在很大程度上保持静止状态，并且与其他组织干细胞一样，被认为依靠糖酵解而不是线粒体OxPHOS来产生ATP2，3。进入细胞周期导致呼吸和OxPHOS增强，导致活性氧（ROS）水平升高，这对HSC的维持和功能有害3。因此，重复的细胞分裂可能导致HSC的自我更新能力降低，并最终导致其耗尽。
在成人造血中，HSC主要经历不对称细胞分裂，在此期间，其中一个子细胞保留HSC潜力并继续依赖糖酵解代谢。另一个子细胞成为原始的祖细胞，失去自我更新能力，但增殖并最终产生分化的功能性造血细胞4。当HSCs分化产生下游祖细胞时，从糖酵解到线粒体代谢的转变被认为是为了提供支持这种快速转变所需的能量和构建块5，正如HSC具有无活性线粒体质量6，7，8，9的观察结果所表明的那样。.相反，线粒体活性（由连锁ROS水平表示）在谱系承诺的祖细胞中远高于HSCs9，10，11。因此，在造血最早阶段发生的代谢变化表明线粒体在调节HSC命运中具有直接和至关重要的作用。
在各种应激条件下（如衰老、癌症、糖尿病和肥胖）下存在的功能失调线粒体12 会干扰 HSC 自我更新能力，通过产生过量的 ROS、损害 ATP 生成和/或改变其他代谢过程来诱导 HSC/祖细胞分化的失衡9，12，13.HSC/祖细胞分化中代谢稳态的扰动可显著影响造血，可能导致血液学异常的发展13.鉴于糖酵解和线粒体OxPHOS对HSC干性和分化的关键影响，研究正常和胁迫条件下的代谢参数是有意义的。通过评估其ECAR和OCR，可以获得对HSC和祖细胞的糖酵解和线粒体功能的宝贵见解，它们分别是细胞糖酵解和线粒体呼吸的指标。
Seahorse细胞外通量分析仪是一种功能强大的仪器，每孔配备两个探针，可同时测量活细胞中的ECAR和OCR，因此可用于实时评估细胞生物能量，以响应各种底物或抑制剂。与分析仪一起使用的检测盒包含进样口，可容纳多达四种药物，以便在测定过程中自动进样。典型的糖酵解压力试验方案如图 1A所示。该测定从测量细胞的ECAR开始，在含有谷氨酰胺但不含有葡萄糖或丙酮酸的糖酵解胁迫测试培养基中孵育。这表示由于细胞的非糖酵解活性而发生的酸化，并且被报道为非糖酵解酸化。接下来以饱和浓度注射葡萄糖。通过糖酵解，细胞中的葡萄糖转化为丙酮酸盐，丙酮酸盐在细胞质中进一步代谢以产生乳酸盐，或在线粒体中产生CO2 和水。
葡萄糖转化为乳酸盐导致净产生并随后将质子释放到细胞外培养基中，导致ECAR14，15，16的快速增加。ECAR中这种葡萄糖刺激的变化被报告为基础条件下的糖酵解。第二次注射由寡霉素（ATP合酶的抑制剂，又名复合物V17）组成，其抑制线粒体ATP的产生。在寡霉素介导的OxPHOS抑制期间，细胞最大限度地上调糖酵解以满足其能量需求。这导致ECAR进一步增加，揭示了细胞的最大糖酵解能力。最大糖酵解能力与基础糖酵解之间的差异称为糖酵解储备。最后，注射2-DG，这导致ECAR显着下降，通常接近非糖酵解酸化水平。2-DG是一种葡萄糖类似物，具有竞争力地与己糖激酶结合，导致糖酵解18的抑制。因此，2-DG诱导的ECAR降低进一步证实糖酵解确实是葡萄糖和寡霉素注射后观察到的ECAR的来源。
图1B 显示了典型线粒体压力测试的示意图。该测定从细胞的基线OCR测量开始，在含有葡萄糖，谷氨酰胺和丙酮酸的线粒体压力测试培养基中孵育。在基础OCR测量之后，在该测定中注入寡霉素，其抑制复合物V，从而减少通过电子传递链（ETC）的电子流动17。因此，OCR在寡霉素注射后降低，OCR的这种降低与线粒体ATP产生有关。第二次注射剂由羰基氰化物-4（三氟甲氧基）苯腙（FCCP），质子团和线粒体OxPHOS17的解偶联剂组成。FCCP通过允许质子流过线粒体内膜来折叠线粒体质子梯度。由于FCCP注射，通过ETC的电子流被抑制，复合物IV以最大水平消耗氧气。最大OCR和基础OCR之间的差异被称为备用呼吸能力，这是细胞在压力条件下对增加的能量需求做出反应的能力的衡量标准。最后，注射两种ETC抑制剂（鱼藤酮，复合物I抑制剂和抗霉素A，复合物III抑制剂17）的混合物，其完全关闭电子流，OCR降低到低水平。鱼藤酮和抗霉素A注射后测量的OCR对应于由细胞内其他过程驱动的非线粒体OCR。非线粒体 OCR 能够计算基底呼吸、质子泄漏和最大呼吸。
基础呼吸代表基线 OCR 和非线粒体 OCR 之间的差异。质子泄漏是指寡霉素注射后的OCR与非线粒体OCR的区别。最大呼吸量表示 FCCP 注射后 OCR 与非线粒体 OCR 之间的差异。耦合效率计算为ATP生产率与基础呼吸速率的百分比。该方法论文提供了使用海马XFe96细胞外通量分析仪测量谱系阴性HSPC中ECAR和OCR的详细方案。该协议描述了分离小鼠谱系阴性HSPC的方法，解释了细胞接种密度和细胞外通量测定中使用的各种药物浓度的优化，并描述了药物治疗策略。
Protocol
Representative Results
Discussion
该方法论文描述了一种优化的方案，用于使用Seahorse细胞外通量分析仪评估小鼠HSPC中的细胞生物能量（糖酵解和OxPHOS）。该装置是一种强大的工具，可同时测量活细胞的ECAR和OCR，分别是糖酵解和线粒体呼吸的指标。因此，它可用于实时评估细胞生物能量学。此外，基于96孔微孔板的平台提供高灵敏度的高通量定量，允许使用单个板同时分析多个样品，而另一种高分辨率呼吸计Oroboros O2k只能同时分?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
伦巴第实验室的工作得到了NIH（NIGMS R01GM101171，NIEHS R21ES032305），DoD（CA190267，CA170628，NF170044和ME200030）和Glenn医学研究基金会的支持。Li实验室的工作由NIH（NHLBI 5R01HL150707）支持。
Materials
| 0.2 μm filter | Corning | 430626 | Used to filter-sterilize the assay media |
| 100 mM sodium pyruvate | Life Technologies | 11360-070 | Component of mitochondrial stress test assay medium |
| 15 mL conical Falcon tubes | Corning | 352096 | Used during HSPCs harvest and to prepare assay drug solutions |
| 200 mM L-glutamine | Life Technologies | 25030-081 | Component of glycolysis stress test and mitochondrial stress test assay media |
| 2-Deoxy-D-glucose (2-DG) | Sigma-Aldrich | D8375 | 3rd drug injection during glycolysis stress test |
| 5x Enrichment buffer (MojoSort) | Biolegend | 480017 | Used for washings during HSPCs harvest |
| Ammonium chloride (NH4Cl) | Fisher Scientific | A661-3 | Component of ACK lysis buffer |
| Antimycin A | Sigma-Aldrich | A8674 | 3rd drug injection during mitochondrial stress test |
| Bio-Rad DC protein assay kit | Bio-Rad | 500-0112 | Used as per manufacturer's instructions |
| Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) | Sigma-Aldrich | C2920 | 2nd drug injection during mitochondrial stress test |
| Cell-Tak | Corning | 354240 | Cell adhesive. Used for coating cell microplates |
| Countes 3 Automated Cell Counter | ThermoFisher Scientific | For cell counting | |
| EDTA | Fisher Scientific | O2793-500 | Component of ACK lysis buffer and RIPA lysis buffer |
| Falcon 70 μm filter | Fisher Scientific | 08-771-2 | Used as cells strainer during HSPCs harvest |
| Gibco Fetal bovine serum (FBS) | Fisher Scientific | 26400044 | Used to prepare assay buffer during HSPCs harvest |
| Gibco HBSS | Fisher Scientific | 14175095 | Used to prepare assay buffer during HSPCs harvest |
| Glucose | Sigma-Aldrich | G7528 | Component of mitochondrial stress test assay medium and first injection of glycolysis stress test |
| Oligomycin | Sigma-Aldrich | O4876 | 2nd drug injection during glycolysis stress test and 1st drug injection during mitochondrial stress test |
| PBS | Life Technologies | 10010-049 | Used to wash cells after assay for protein quantification |
| Potassium bicarbonate (KHCO3) | Fisher Scientific | P235-500 | Component of ACK lysis buffer |
| Protease Inhibitor Cocktail (PIC) | Roche | 11836170001 | Supplied as tablets. One tablet was dissolved in 10 mL of RIPA buffer to make 1x PIC. |
| Rat biotin antimouse-B220, Clone ID: RA3-6B2 | Biolegend | 103203 | Used for lineage depletion during HSPCs harvest |
| Rat biotin antimouse-CD2, Clone ID: RM2-5 | Biolegend | 100103 | Used for lineage depletion during HSPCs harvest |
| Rat biotin antimouse-CD3, Clone ID: 17A2 | Biolegend | 100243 | Used for lineage depletion during HSPCs harvest |
| Rat biotin antimouse-CD5, Clone ID: 53-7.3 | Biolegend | 100603 | Used for lineage depletion during HSPCs harvest |
| Rat biotin antimouse-CD8, Clone ID: 53-6.7 | Biolegend | 100703 | Used for lineage depletion during HSPCs harvest |
| Rat biotin antimouse-Gr-1, Clone ID: RB6-8C5 | Biolegend | 108403 | Used for lineage depletion during HSPCs harvest |
| Rat biotin antimouse-Ter-119, Clone ID: TER-119 | Biolegend | 116203 | Used for lineage depletion during HSPCs harvest |
| Rotenone | Sigma-Aldrich | R8875 | 3rd drug injection during mitochondrial stress test |
| Seahorse XFe96 extracellular flux analyzer | Seahorse Biosciences now Agilent | For ECAR and OCR measurments in real time. | |
| Sodium bicarbonate | Sigma-Aldrich | S5761 | Used to make Cell-adhesive solution for microplate coating |
| Sodium chloride (NaCl) | Fisher | BP358 | Component of RIPA lysis buffer |
| Sodium deoxycholate | Sigma-Aldrich | D6750 | Component of RIPA lysis buffer |
| Sodium Fluoride (NaF) | Sigma-Aldrich | S7920 | Component of RIPA lysis buffer |
| Sodium hydroxide (NaOH) | Sigma-Aldrich | S8045 | Prepared 1 N solution. Used for pH normalization |
| Streptavidin Nanobeads (MojoSort) | Biolegend | 480015 | Used for lineage depletion during HSPCs harvest |
| Tris-HCl | Fisher | BP153 | Component of RIPA lysis buffer |
| XF base medium | Agilent | 102353-100 | base medium used to prepare glycolysis stress test and mitochondrial stress test assay media |
| XF prep station | Seahorse Biosciences | Used for non-CO2 37 °C incubations | |
| XFe96 extracellular FluxPak | Agilent | 102416-100 or 102601-100 | Includes assay cartridges with utility plates, loading guide flats for loading drugs onto the assay cartridge, XF calibrant solution, and XF cell culture microplate |
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