木尿造血干细胞和祖细胞和MLL-AF9驱动白血病线粒体反应氧物种的流细胞学分析
Summary
我们描述了一种使用多参数流细胞测定法来检测小鼠健康造血干细胞和祖细胞（HSPCs）中的线粒体活性氧物种（ROS）和白血病细胞的方法，这些细胞来自急性骨髓性白血病（AML）的小鼠模型。MLL-AF9。
Abstract
我们提出了一种流式细胞学方法，用于分析健康小鼠以及MLL-AF9驱动的AML小鼠的各种活骨髓（BM）衍生干细胞和祖细胞群中的线粒体ROS。具体来说，我们描述了一个两步细胞染色过程，其中健康或白血病BM细胞首先用荧光染料染色，检测线粒体超氧化物，然后染色与氟铬链接单克隆抗体使用区分各种健康和恶性造血祖群。我们还提供了一个通过流式细胞测定采集和分析样品的策略。整个协议可以在3-4小时的时间内执行。我们还强调了要考虑的关键变量，以及监测活造血干细胞和白血病茎线粒体中ROS生产的优点和局限性，并使用流式细胞测定的氟原染料进行祖生子种群.此外，我们提供的数据，线粒体ROS丰度在不同的健康HSPC亚群体和白血病祖细胞之间变化，并讨论了该技术在血液学研究的可能应用。
Introduction
活性氧物种（ROS）是从分子氧中提取的高度活性分子。ROS生产最明确的细胞位置是线粒体，在氧化磷酸化（OXPHOS）期间通过电子传输链（ETC）的电子被分子氧吸收，从而形成特定的ROS类型称为超氧化物1。通过一系列酶（称为超氧化物歧化酶或SOD）的作用，超氧化物转化为过氧化氢，随后通过催化酶或谷胱甘肽过氧化物酶（GPX）等酶中和成水。ROS 调节机制中的扰动可导致 ROS 的过量生产，通常称为氧化应激，具有有害且潜在的致命细胞后果，如大分子损伤（即 DNA、蛋白质、脂质）。此外，氧化应激与多种疾病有关，如糖尿病、炎症性疾病、衰老和肿瘤2、3、4。为了保持氧化平衡和防止氧化应激，细胞拥有各种ROS调节机制5。
某些ROS的生理水平是必要的适当的胚胎和成人造核6。然而，过量的ROS与DNA损伤、细胞分化和造血干细胞和祖细胞池的衰竭有关。也有证据显示，在白血病和健康细胞之间，氧化还原生物学的改变可能有所不同。例如，在急性骨髓性白血病（AML）细胞中，ROS水平往往高于其健康细胞，其他研究表明，白血病干细胞维持低稳态的ROS水平，以生存7，8。重要的是，利用这些氧化还原差异的治疗策略在几个人类癌症环境中显示出了希望9，10。因此，允许评估小鼠模型中ROS水平的检测可以增进我们对这些物种如何促进细胞生理学和疾病发病机制的理解，并可能为评估新型氧化还原靶向抗癌疗法。
Protocol
本议定书中描述的所有动物程序均已获得福克斯大通癌症中心机构动物护理和使用委员会（IACUC）的批准。 注：协议工作流程分为4个部分，如图1所示，所需试剂列在材料表中。 1. 骨髓 （BM） 隔离 注：MLL-AF9白血病小鼠的产生，如前11所述。 如前<su…
Representative Results
提出了一种分析多个健康型和MLL-AF9表达性白血病祖体群线粒体ROS的方法。图 1显示了协议工作流的原理图视图，该视图由 4 个主要步骤组成：1） BM 与小鼠分离;2） 用能识别线粒体ROS的荧光染料染色BM细胞，特别是超氧化物;3） 表面标记抗体染色，以划定各种健康和白血病造血人群;4） 流式细胞学采集和分析。 图 2A，B?…
Discussion
为检测ROS而开发的荧光染料，经常通过显微镜或流式细胞22在固定细胞中进行评估。使用线粒体ROS氟致染料对BM细胞中线粒体ROS进行流式细胞测量具有两个主要优点：1）它是一种快速简便的技术，适合活细胞分析;2）它允许区分和分析罕见使用表面标记染色在BM的单细胞水平上。此处介绍的分步协议旨在研究两只健康小鼠的造血干细胞和祖体种群的活体氧化还原状态，以及?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了福克斯大通癌症中心董事会（DDM）、美国血液学学会学者奖（SMS）、美国癌症协会RSG（SMS）和国防部的支持（奖状：W81XWH-18-1-0472）。
Materials
| Heat inactivated FBS | VWR Seradigm LIFE SCIENCE | 97068-085 | Media |
| Penicillin Streptomycin | Corning | 30-002-CI | Media |
| PBS | Fisher Scientific | BP399-20 | Buffer |
| 15 mL conical tube | BD falcon | 352096 | Tissue Culture Supplies |
| 50 mL conical tube | BD falcon | 352098 | Tissue Culture Supplies |
| 40 μm cell strainers | Fisher Scientific | 22-363-547 | Tissue Culture Supplies |
| RBC Lysis Buffer | Fisher Scientific | 50-112-9751 | Tissue Culture Supplies |
| Menadione sodium Bisulfite | Sigma aldrich | M5750 | Pro-oxidant |
| NAC | Sigma aldrich | A7250 | Anti-oxidant |
| CD3 PE-Cy5 clone 145-2c11 | Biolegend | 100310 | Antibody |
| CD4 PE-Cy5 clone RM4-5 | eBioscience | 15-0041-81 | Antibody |
| CD8 PE-Cy5 clone 53-6.7 | eBioscience | 15-0081-81 | Antibody |
| CD19 PE-Cy5 clone 6D5 | Biolegend | 115510 | Antibody |
| B220 PE-Cy5 clone RA3-6B2 | Biolegend | 103210 | Antibody |
| Gr1 PE-Cy5 clone RB6-8C5 | Biolegend | 108410 | Antibody |
| Ter119 PE-Cy5 clone Ter-119 | Biolegend | 116210 | Antibody |
| CD48 PE-Cy5 clone HM48-1 | Biolegend | 103420 | Antibody |
| CD117 APC-Cy7 clone 2B8 | Biolegend | 105825 | Antibody |
| Sca1 peacific Blue clone D7 | Biolegend | 108120 | Antibody |
| CD150 APC clone TC15-12F12.2 | Biolegend | 115909 | Antibody |
| CD34 FITC clone RAM34 | BD Bioscience | 553733 | Antibody |
| CD45.2 APC clone 104 | Biolegend | 1098313 | Antibody |
| MitoSOX Red | ThermoFisher Scientific | M36008 | Dye |
| Mitotracker Green | ThermoFisher Scientific | M7514 | Dye |
| Live/dead Yellow Dye | ThermoFisher Scientific | L34967 | Dye |
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Citar este artigo
Di Marcantonio, D., Sykes, S. M. Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia. J. Vis. Exp. (151), e59593, doi:10.3791/59593 (2019).