Name: two flow cytometric approaches of nkg2d ligand surface detection to distinguish stem cells from bulk subpopulations in acute myeloid leukemia
Uploaded: 2021-02-21T13:00:00
Description: Watch this Scientific Journal Video about Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia at JoVE.com

这项研究得到了瑞士国家科学基金会（179239）、抗癌基金会（Zuerich）、威廉·桑德基金会和CL（2019.042.1）以及诺华医学-生物研究基金会的资助。此外，该项目还根据玛丽·斯考多夫斯卡-库里第765104号赠款协议，从欧洲联盟的Horizo 2020研究和创新项目获得资金。我们感谢巴塞尔的流细胞学设施的支持。

患者样本是在巴塞尔和图本根大学医院伦理审查委员会批准后采集的。
1. NKG2D融合蛋白的生物酸化
注：此步骤根据制造商的说明，用生物素化套件（参见 材料表）执行。协议的此步骤必须在染色前至少执行 24 小时。生物素化 NKG2D 融合蛋白应存储在 -20 °C 下。
<ol><li>解冻生物素，以及NKG2D聚变蛋白管在室温下（RT）。 注：如果细胞需要无?...

<ol>
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在这里，我们提出了两种流细胞测量方法，可以检测人类原体AML细胞上的NKG2DL表面表达。我们表明，这两种检测方法都可以与其他抗体染色（例如检测 CD34 表达式）配合使用。类似的染色也可以在其他主要细胞类型和细胞系上执行。
我们最近表明，在AML患者爆炸表面没有NKG2DL可以丰富LSC14。在 AML 中，NKG2DL 阴性但非 NKG2DL 阳性白血病亚聚众具有克隆和 体?...

NK细胞是先天免疫系统的重要影响者，可以识别和消除恶性细胞或有压力的健康细胞（例如，通过病毒感染），而无需事先刺激抗原1。这个过程通过激活受体的复杂剧目（如天然细胞毒性受体 （NCR）、NKG2D 和 CD16） 以及主要由杀手免疫球蛋白样受体 （KIR）2表示的抑制受体进行严格调控。KIR 与人体白细胞抗原 （HLA） I 类分子结合在体细胞上，确保自我识别并传达 NK 细胞耐受性。另一方面，缺乏自我识别和激活受体对目标细胞的配体的结合增加，导致细胞毒性颗粒的释放，导致NK细胞介导细胞毒性1。最后，NK细胞可以通过将激活受体CD16与表示Ig （FcR）2Fc 部分的目标结合来发挥抗体依赖性细胞毒性 （ADCC） 的作用。除了直接细胞毒性，NK细胞还可以触发细胞因子释放桥接与自适应免疫系统3与先天。
NKG2D 是 NK、NKT、+T 和天真的 CD8+ T 细胞4上表达的主要激活受体，使此类细胞毒性免疫细胞能够识别和解解 NKG2D 配体 （NKG2DL） 表达目标细胞。健康细胞通常不表达 NKG2DL。相反，NKG2DL表达是对恶性或病毒感染细胞的调节，使这些适应免疫清除5。
人类NKG2DL家族由8个已知分子组成，其中两个MHC I链相关分子A和B（MICA和MICB6）和巨细胞病毒UL16结合蛋白1/6（ULBP1-67）。NKG2DL 的表达在转录、转录后以及后翻译级别8上进行调节。因此，虽然NKG2DL表达在健康细胞表面通常无法检测到，但NKG2DL mRNA9和细胞内蛋白质表达在健康组织中报告。这种表达的功能相关性和这种不同表达模式背后的机制仍有待界定。
癌细胞中NKG2DL表达的机械调控是一个引人入胜的调查领域。已知与细胞应激（如热冲击应激通路9）或 DNA 损伤相关通路（如厌食性泰兰吉拉西亚变异 （ATM） 和 Rad3 相关 （ATR） 通路11）以及病毒或细菌感染有关的通路与 NKG2DL 表达12的诱导直接相关。然而，即使NKG2DL的表面表达已被有效地诱导，这种表达可以通过原解剖介质脱落再次丢失，这种机制与免疫逃生和一些癌症的临床预后差有关。
细胞表面 NKG2DL 的缺失也可能对 AML 患者发挥重要作用。在这里，用强化化疗治疗通常诱导缓解，但复发通常发生在白血病干细胞（LSC），选择性地生存化疗和逃避免疫反应。例如，正如我们最近所表明的，LSC通过抑制NK2DL表面表达14来逃避NK细胞裂解。
相反，没有表面NKG2DL表达可以作为一种方法来识别和恶毒地分离假定干细胞状细胞的亚群从散装对应白血病亚群。在这里，我们提出了两种流细胞学方法，可用于检测 NKG2DL 表面表达，从而识别白血病中的 NKG2DL 阴性干细胞，也许还可以用于其他癌症：泛配体表面识别方法和涉及单个或池状抗体染色的方法，以识别单个已知 NKG2DL 蛋白质。

<table>
	<tbody>
		<tr>
			<td>7-amminoactinomycin D (7-AAD)</td>
			<td>Invitrogen</td>
			<td>A1310</td>
			<td>Viability dye</td>
		</tr>
		<tr>
			<td>96 well plate U bottom</td>
			<td>Sarstedt</td>
			<td>833925500</td>
			<td>96 Well plate for our Flow cytometer</td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human cd34</td>
			<td>BD</td>
			<td>555824</td>
			<td>Antibody detecting CD34 
			RRID: AB_398614</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>PanReac AppliChem</td>
			<td>A1391,0050</td>
			<td>Component of the staining buffer</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Roth</td>
			<td>8043.1</td>
			<td>Component of the staining buffer</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>BioConcept</td>
			<td>2-01F10-I</td>
			<td>Component of the supplemented RPMI medium</td>
		</tr>
		<tr>
			<td>FlowJo 10.2</td>
			<td>BD</td>
			<td>/</td>
			<td>Software enabling data analysis for flow cytometry experiment</td>
		</tr>
		<tr>
			<td>Goat- anti-Rabbit IgG (H+L) Alexa Fluor 488</td>
			<td>Thermo Scientific</td>
			<td>A21222</td>
			<td>Secondary antibody detecting the primary antibodies for MICA and MICB 
			RRID: AB_1037853</td>
		</tr>
		<tr>
			<td>Human NKG2D Fc Chimera Protein, CF</td>
			<td>R&#38;D</td>
			<td>1299-NK-050</td>
			<td>Fusion Protein detecting all NKG2DLs 
			RRID:</td>
		</tr>
		<tr>
			<td>Human ULBP-1 Antibody</td>
			<td>R&#38;D</td>
			<td>AF1380</td>
			<td>Antibody detecting ULBP1 
			RRID: AB_354765</td>
		</tr>
		<tr>
			<td>Human ULBP-2/5/6 Antibody</td>
			<td>R&#38;D</td>
			<td>AF1298</td>
			<td>Antibody detecting ULBP2/5/6 
			RRID: AB_354725</td>
		</tr>
		<tr>
			<td>Human ULBP-3 Antibody</td>
			<td>R&#38;D</td>
			<td>AF1517</td>
			<td>Antibody detecting ULBP3 
			RRID: AB_354835</td>
		</tr>
		<tr>
			<td>MICA Polyclonal Antibody</td>
			<td>Thermo Scientific</td>
			<td>PA5-35346</td>
			<td>Antibody detecting MICA 
			RRID: AB_2552656</td>
		</tr>
		<tr>
			<td>MICB Polyclonal Antibody</td>
			<td>Thermo Scientific</td>
			<td>PA5-66698</td>
			<td>Antibody detecting MICB 
			RRID: AB_2663413</td>
		</tr>
		<tr>
			<td>One-step Antibody Biotinylation Kit 1 strip, for 8 reactions</td>
			<td>Miltenyibiotec</td>
			<td>130-093-385</td>
			<td>Biotinylation kit for the NKG2DL fusion protein</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537-500ML</td>
			<td>Component of the staining buffer</td>
		</tr>
		<tr>
			<td>Rabbit anti-Goat IgG (H+L) Alexa Fluor 488</td>
			<td>Thermo Scientific</td>
			<td>A11034</td>
			<td>Secondary antibody detecting the primary antibodies for the ULBPs 
			RRID: AB_2576217</td>
		</tr>
		<tr>
			<td>Rainbow Calibration Particles (8-peaks) 3.0 um</td>
			<td>Spherotech Inc.</td>
			<td>RCP-30-20A</td>
			<td>Beads used for flow cytometry device maintainance</td>
		</tr>
		<tr>
			<td>RPMI medium</td>
			<td>Sigma Aldrich</td>
			<td>R8758-500ML</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>RayBright Universal Compensation Beads</td>
			<td>Raybiotech</td>
			<td>137-00013-100</td>
			<td>Beads used to create the compensation matrix</td>
		</tr>
		<tr>
			<td>Streptavidin, R-Phycoerythrin Conjugate (SAPE) - 1 mg/mL</td>
			<td>Invitrogen</td>
			<td>S866</td>
			<td>Seondary step for the biotinylated NKG2DL fusion protein detection</td>
		</tr>
	</tbody>
</table>

two flow cytometric approaches of nkg2d ligand surface detection to distinguish stem cells from bulk subpopulations in acute myeloid leukemia

在同一患者中，NKG2D配体（NKG2DL）表面表达的缺失被证明能够区分具有干细胞特性的白血病亚群（所谓的白血病干细胞，LSC）和更差异化的对应白血病细胞，这些白血病细胞虽然携带类似的白血病特异性基因突变，但缺乏疾病启动潜力。NKG2DL 是生化高度多样化的 MHC 类 I 类自分子。在家居静止条件下的健康细胞通常不会在细胞表面表达 NKG2DL。相反，这些配体的表达是在接触细胞应激（如致癌转化或传染性刺激）时诱发的，通过NKG2D受体表达免疫细胞（如自然杀手（NK）细胞，通过裂解触发受损细胞的消除。有趣的是，NKG2DL表面表达在LSC亚聚众中被选择性地抑制，使这些细胞能够逃避NKG2D介质的免疫监测。在这里，我们提出了两种不同的流细胞学方法的并排分析，允许对癌细胞上的 NKG2DL 表面表达进行调查，即涉及泛配体识别的方法和涉及对单个配体的多种抗体染色的方法。这些方法可用于将具有假定癌症干细胞特性的可行 NKG2DL 负细胞亚聚众与 NKG2DL 阳性非 LSC 分离。

这里提出的两个协议都允许使用CD34（LSC17的已知标记）的流细胞分析来丰富AML LSC，同时利用泛配体识别或与单个配体的组合抗体进行染色，从而与NKG2DL表面表达相结合。在图1中，我们显示分析的AML样本对CD34和NKG2DL呈阳性反应，但也存在阴性亚群，这表现在总共有四个不同的种群。我们的数据显示了这种染色的典型盖子策略，从通过 FSC 和 SSC 选择细胞的主...

Watch this Scientific Journal Video about Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia at JoVE.com

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

我们在人类原发性急性骨髓性白血病 （AML） 样本中提出了 NKG2D 配体 （NKG2DL） 检测的两种不同的染色方案。第一种方法基于融合蛋白，能够识别所有已知和潜在的未知配体，而第二种方法依赖于添加多个抗NKG2DL抗体。

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 ...

此协议显示了检测 AML 细胞表面 NKG2DL 的两种不同方法。该技术提供了一种快速、用户友好的染色方法，可检测所有已知的、可能未知的 NKG2DL。这种方法允许将白血病干细胞与大宗AML细胞分离，从而有可能进一步描述这些细胞的特征。首先在室温下解冻生物素和NKG2D融合蛋白管。快速旋转 NKG2D FC 粉末，然后加入 500 微升 PBS 来重组粉末，然后使用 P-1000 微管进行彻底混合。将 NKG2DL 融合蛋白的 100 微升添加到生物素管中，以获得每毫升 10 微克的最终浓度，并将溶液与 P-100 微管彻底混合。要解冻 AML 细胞，请从液氮储存中取出含有原位 AML 的低温，并立即将其放入 37 摄氏度的水浴中。轻轻地在水中来回移动管子，让小瓶内的内容解冻，直到只剩下一个小冰晶。立即将解冻的细胞转移到含有管子的介质中，然后用一毫升的介质冲洗小瓶。将细胞以300倍G离心10分钟，并丢弃超高分子，而不会干扰颗粒。用含有10%FCS的5毫升RPMI介质清洗细胞，并重复离心。用染色缓冲器将细胞颗粒重新使用，最终浓度为每毫升0.5倍至7个细胞。然后将细胞悬浮的 100 微升转移到细胞培养 96 井 U 底板，并以 300 倍 G 离心板 10 分钟。丢弃超高纳特而不干扰颗粒。准备生物素化 NKG2D 融合蛋白的主混合物，使细胞以每口井 50 微升的最终体积进行再利用，最终 NKG2D 浓度为每口井每毫升 10 微克。加入准备好的主混合物，用200微升移液器重新使用细胞颗粒。如文本手稿中所述，孵化和离心板。使用链球菌素 PE 准备主组合，以便将细胞重新悬念在最终体积为 50 微升。将主混合物添加到细胞中，然后用 300 微升多通道移液器重新使用颗粒。离心板并丢弃超高纳特后，使用 300 微升多通道微管将细胞颗粒重新用于 200 微升染色缓冲器加 7-AAD。然后使用流细胞学设备分析细胞。分析的 AML 样本对 CD34 和 NKG2DL 呈阳性，但也存在阴性亚群，总共有四个不同的群体。典型的生长策略从通过 FSC 和 SSC 选择细胞的主要群体开始。下游分析中排除了双细胞和死细胞。使用 FMO 控制调整闸门，以确保正确识别正细胞。此处突出显示了 CD34 与 NKG2DL 的细胞阳性的荧光强度。对 CD34 呈阳性的 AML 细胞显示 NKG2DL 的较低表面表达，表明 NKG2DL 表达与缺乏干细胞有关。三个原发性AML样本显示融合蛋白染色阳性事件为19.8、49.8和89.4%，而聚合抗NKG2DL抗体染色为20.4、50.4和90.6%。另一方面，单个配体染色显示一系列阳性事件高达 92%，百分比因配体而异。在尝试此方法时，解冻主要 AML 细胞时快速解冻非常重要，因为它们很脆弱，在步骤中很容易死亡。执行此协议后，可以执行基于 NKG2DL 信号的细胞排序，以进一步调查该群。

Research

JoVE Journal

Cancer Research

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 ...

科研

癌症研究

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

离心淘洗制备不同细胞周期的原发性急性淋巴细胞白血病细胞

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

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 ...

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 ...

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

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 ...

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

实时检测和捕获共培养物中的侵袭细胞亚群

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

腹膜内移植在小鼠中产生急性髓系白血病

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...

使用非放射性化学发光检测定量端粒长度

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

作者聚焦：TAGGG 端粒长度测定性能参数的优化  

TAGGG端粒长度测定性能参数优化

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...