Name: flow cytometric analysis of mitochondrial reactive oxygen species in murine hematopoietic stem and progenitor cells and mll-af9 driven leukemia
Uploaded: 2019-09-05T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia at JoVE.com

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）。

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。
<ol><li>如前<su...

<ol>
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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?...

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。

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			<td height="40" style="height:40px;width:247px;">Heat inactivated FBS</td>
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			<td>Corning</td>
			<td>30-002-CI</td>
			<td>Media</td>
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		<tr height="40">
			<td height="40" style="height:40px;">PBS</td>
			<td>Fisher Scientific</td>
			<td>BP399-20</td>
			<td>Buffer</td>
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		<tr height="40">
			<td height="40" style="height:40px;">15 mL conical tube</td>
			<td>BD falcon</td>
			<td>352096</td>
			<td>Tissue Culture Supplies</td>
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		<tr height="40">
			<td height="40" style="height:40px;">50 mL conical tube</td>
			<td>BD falcon</td>
			<td>352098</td>
			<td>Tissue Culture Supplies</td>
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			<td height="40" style="height:40px;">40 &#956;m cell strainers</td>
			<td>Fisher Scientific</td>
			<td>22-363-547</td>
			<td>Tissue Culture Supplies</td>
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			<td height="40" style="height:40px;">RBC Lysis Buffer</td>
			<td>Fisher Scientific</td>
			<td>50-112-9751</td>
			<td>Tissue Culture Supplies</td>
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			<td height="40" style="height:40px;">Menadione sodium Bisulfite</td>
			<td>Sigma aldrich</td>
			<td>M5750</td>
			<td>Pro-oxidant</td>
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		<tr height="40">
			<td height="40" style="height:40px;">NAC</td>
			<td>Sigma aldrich</td>
			<td>A7250</td>
			<td>Anti-oxidant</td>
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			<td height="42" style="height:42px;width:247px;">CD3 PE-Cy5 clone 145-2c11</td>
			<td>Biolegend</td>
			<td>100310</td>
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			<td height="42" style="height:42px;width:247px;">CD4 PE-Cy5 clone RM4-5</td>
			<td>eBioscience</td>
			<td>15-0041-81</td>
			<td>Antibody</td>
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			<td height="42" style="height:42px;width:247px;">CD8 PE-Cy5 clone 53-6.7</td>
			<td>eBioscience</td>
			<td>15-0081-81</td>
			<td>Antibody</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:247px;">CD19 PE-Cy5 clone 6D5</td>
			<td>Biolegend</td>
			<td>115510</td>
			<td>Antibody</td>
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			<td height="42" style="height:42px;width:247px;">B220 PE-Cy5 clone RA3-6B2</td>
			<td>Biolegend</td>
			<td>103210</td>
			<td>Antibody</td>
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			<td height="42" style="height:42px;width:247px;">Gr1 PE-Cy5 clone RB6-8C5</td>
			<td>Biolegend</td>
			<td>108410</td>
			<td>Antibody</td>
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			<td height="40" style="height:40px;">Ter119 PE-Cy5 clone Ter-119</td>
			<td>Biolegend</td>
			<td>116210</td>
			<td>Antibody</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CD48 PE-Cy5 clone HM48-1</td>
			<td>Biolegend</td>
			<td>103420</td>
			<td>Antibody</td>
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		<tr height="40">
			<td height="40" style="height:40px;">CD117&#160; APC-Cy7 clone 2B8</td>
			<td>Biolegend</td>
			<td>105825</td>
			<td>Antibody</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Sca1 peacific Blue clone D7</td>
			<td>Biolegend</td>
			<td>108120</td>
			<td>Antibody</td>
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		<tr height="40">
			<td height="40" style="height:40px;">CD150 APC clone TC15-12F12.2</td>
			<td>Biolegend</td>
			<td>115909</td>
			<td>Antibody</td>
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		<tr height="40">
			<td height="40" style="height:40px;">CD34 FITC clone RAM34</td>
			<td>BD Bioscience</td>
			<td>553733</td>
			<td>Antibody</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CD45.2 APC clone 104</td>
			<td>Biolegend</td>
			<td>1098313</td>
			<td>Antibody</td>
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		<tr height="40">
			<td height="40" style="height:40px;">MitoSOX Red</td>
			<td>ThermoFisher Scientific</td>
			<td>M36008</td>
			<td>Dye</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mitotracker Green</td>
			<td>ThermoFisher Scientific</td>
			<td>M7514</td>
			<td>Dye</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Live/dead Yellow Dye</td>
			<td>ThermoFisher Scientific</td>
			<td>L34967</td>
			<td>Dye</td>
		</tr>
	</tbody>
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flow cytometric analysis of mitochondrial reactive oxygen species in murine hematopoietic stem and progenitor cells and mll-af9 driven leukemia

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。

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?...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia at JoVE.com

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

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。

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

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

活性氧物种或ROS，是由细胞产生的高活性氧物种，影响它们的行为。虽然过量的ROS也会导致广泛的细胞破坏，在严重的情况下，细胞死亡。因此，维持ROS平衡对细胞功能和生存至关重要。此处提出的协议侧重于测量由线粒体生成的特定类型的 ROS，主要作为活体、正常或健康的造血干细胞和祖细胞群中的代谢副产物，以及来自血癌、急性骨髓性白血病或 AML 小鼠模型的白血病细胞。该协议还可用于评估基因操作，如基因缺失或过度表达，如何影响线粒体ROS在几个健康和恶性造血种群。因此，可能提供有关重做状态和细胞代谢的有见地的信息。该技术授权对原生探针进行流结计量分析，以监测活体恶性造血干细胞和祖细胞子群中的线粒体ROS生产。该协议非常简单，可以在几个小时内完成。此外，它适用于活细胞分析，并允许区分和分析线粒体ROS在干细胞群体在骨髓，使用染色的表面标记。在从野生紧绷小鼠和白血病MLL-AF9小鼠身上采集单核骨髓细胞后，根据手稿，将200微升细胞悬浮到9个单色控制管中。将剩余的细胞分成其他实验管，每个细胞200微升。然后放入离心机中，分别放置两个包含骨髓细胞和白血病细胞的实验管，以及一个控制管。在300次G下离心5分钟。接下来，根据制造商的说明，将上一代细胞去掉，并在室温 F-PBS 中用活细胞/死细胞污渍重新悬浮。在冰上孵育30分钟。之后，在三个活/死染色管中加入1毫升室温F-PBS，以及一个单一颜色控制管，用于线粒体ROS染料染色。在室温下，以 300 次 G 将管子放入离心机中，5 分钟。然后，通过重新悬浮50微克的线粒体ROS染料在13微升的DMSO中，获得5毫摩尔线粒体ROS染料库存溶液。然后将13毫升室温F-PBS加入13微升线粒体ROS染料中，稀释至5微摩尔的最终浓度。如有必要，在溶液中加入13微升50毫摩尔维拉帕米尔，以抑制线粒体外流泵。从离心机获取管子，吸走活细胞/死细胞污渍的洗涤。在活/死染色控制中加入 200 微升 F-PBS，并将其留在冰中，直到准备好开始分析。在每个实验管中加入200微升含有Verapamil的线粒体ROS染料染色，以及线粒体ROS染色控制管。涡流在黑暗中37摄氏度下混合和孵育10分钟。然后向控制和实验管中加入1毫升室温F-PBS。在室温下以 300 倍 G 离心 5 分钟。吸走超吸液，再用一毫升室温F-PBS清洗细胞。在室温下，在300倍G下再次离心5分钟。首先，为健康和白血病骨髓细胞准备两种抗体鸡尾酒。从含有健康骨髓细胞的实验管中吸出上流液，并在管中加入200微升的抗体鸡尾酒一号。漩涡混合。还要通过添加200微升F-PBS和一微升的相应抗体来准备单色控制管。在黑暗中的冰上孵育60分钟。从含有白血病骨髓的实验管中吸出上流液，并在管中加入200微升的抗体鸡尾酒二号。漩涡混合。在黑暗中的冰上孵育60分钟。之后，用1毫升的冷F-PBS清洗所有管子，并在室温下以300倍G的离心机清洗5分钟。将细胞重新悬浮在500微升的冷F-PBS中。使用 40 微米过滤器将细胞悬浮液转移到每个流动细胞计管中，以排除聚集体。首先，将一根无污渍控制管插入流式细胞仪，开始采集以补偿流式细胞仪。对其他控制管重复。之后，读取实验管以设置闸门。要分析细胞群的大小和复杂性，首先为健康的HSPC设置前进散射面积和侧散射区图图群。按方形门按钮，从前进和侧面散射图中清除无关的碎屑。然后，在向前散射区域和高度图上，使用双鉴别器将双精度点分门出。选择活细胞、血统低细胞和各种健康的HSPC。对于每个感兴趣的群体，分析组织图图中TRPE通道的中位荧光强度，以评估线粒体ROS信号的差异。对白血病人群重复大小分析、浇注和直方图绘制相同的过程。从健康小鼠身上分离出的骨髓细胞被染色为活/死染料和线粒体ROS染料，随后染色的抗体识别血统标记：加CD48、C-kit、Sca-1、CD34和CD150。此外，白血病小鼠的骨髓细胞也沾染了CD45.2，以区分MLL-AF9白血病细胞与沾有CD45.1的健康接受者骨髓细胞。对健康的CD48阴性LSK和骨髓祖体之间的线粒体ROS染色的比较表明，骨髓祖体表现出明显高于线粒体ROS染色水平。此外，与c-Kit中低白血病细胞、健康CD48阴性LSK和骨髓祖细胞相比，c-Kit高白血病祖细胞的线粒体ROS染色水平显著提高。C-Kit中低性白血病细胞，也显示比CD48阴性LSK细胞，但不是骨髓祖细胞的显著值。线粒体 ROS 染料具有高度反应性，需要适当的洗涤步骤，以确保在开始以下步骤之前去除任何多余的染料。有几个化学上不同的ROS氟原染料，可用于本协议，以实现一点知识的重性状态在活造血细胞。这项技术在文献中得到了广泛的应用，如果与正常细胞相比，可以提供有关在白血病细胞中不同调节的代谢和信号通路有用的见解。

Research

JoVE Journal

Cancer Research

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

详细的定性C1498小鼠细胞系及其关联白血病小鼠模型协议

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

科研

癌症研究

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

测定急性髓系白血病可测残留病和白血病干细胞的骨髓标本和过程综合协议

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

流式细胞仪估测原发性急性髓系白血病和患者源性移植中白血病干细胞的诊断和随访

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation

慢性淋巴细胞白血病免疫球蛋白基因序列分析: 从患者材料到序列解释

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

原发性白血病细胞代谢谱的评价

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

突变黑色素细胞干细胞引发小鼠黑色素瘤的时空控制

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

在结肠直肠癌细胞的三维球体培养物中使用无细胞益生菌超常剂评估细胞死亡

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

NKG2D 利甘德表面检测的两种流细胞学方法，以区分干细胞与急性骨髓性白血病中的散装亚群

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

放线菌素D处理SiHa宫颈癌细胞中凋亡生物标志物的流式细胞术分析

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...