Name: live-3d-cell immunocytochemistry assays of pediatric diffuse midline glioma
Uploaded: 2021-11-11T13:00:00
Description: Watch this Scientific Journal Video about Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma at JoVE.com

我们要感谢Silvia Soddu博士和Giulia Federici博士（意大利罗马IRCCS-Regina Elena国家癌症研究所细胞网络和分子治疗靶点部门）在成像协议的初步设置中使用IncuCyte S3活细胞成像系统。此外，我们感谢Bernadett Kolozsvari的技术建议。该研究得到了英国癌症儿童拨款（16-234）和意大利卫生部Ricerca Corrente的支持。M Vinci是英国癌症儿童研究员。R Ferretti是Veronesi基金会奖学金（2018年和2019年）的获得者。作者感谢Fondazione Heal的支持，并感谢儿童医院基金会为昆士兰儿童肿瘤银行提供资金。

该协议遵循机构人类研究伦理委员会的指导方针。
注意：本研究使用Incucyte S3和/或SX5活细胞分析仪器（参考为活细胞分析仪器）进行。
1. 产生可重复大小的肿瘤球体
注意：Vinci 等人 2015 7,12 描述的协议（第 1 节）如下所述，并进行了一些修改：
<ol><li>收集DMG H3K27M突变神经球?...

<ol>
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我们在这里采用的用于儿科DMG侵袭和迁移的活3D细胞免疫细胞化学也可以很容易地适应其他高度侵袭的肿瘤细胞类型，包括乳腺癌和结肠癌细胞系。 
与以前进行的活2D细胞免疫细胞化学测定10不同，在3D中工作时，建议注意一些关键步骤。特别是，对于我们描述的侵袭测定，建议在添加BMM之前，将ALR /抗体混合物直接添加到每个孔中具有NS的培养基中，而不是?...

肿瘤细胞逃避和通过周围组织传播的能力是癌症的标志1。特别是，肿瘤细胞运动是恶性肿瘤的特征，无论是转移性肿瘤类型如乳腺癌2或结直肠癌3，还是局部侵袭性类型如弥漫性胶质瘤4,5。
成像在肿瘤细胞表型的许多方面研究中具有核心作用;然而，在研究动态细胞过程（例如迁移和侵袭）时，当形态和细胞间相互作用发生变化时，活细胞成像绝对是首选 6,7 并且随着时间的推移可以更容易地检查。对于活细胞成像，可以使用不同的光学显微镜系统，从相衬到共聚焦荧光显微镜，并在配备温度和CO2控制室的倒置显微镜上进行短时间或长时间的图像采集，或在具有内置腔室的高内涵图像分析系统中，或者可以在可以坐在培养箱中的图像系统中，而无需在整个过程中干扰细胞的实验。所用系统的选择通常取决于许多因素，例如所需的分辨率、总采集时间和时间间隔的长度、使用的容器类型和测定的通量（单室或多孔板）、所用细胞（珍贵和/或稀有细胞）的灵敏度以及细胞的光毒性（如果存在荧光团）。
关于活模式下的荧光成像，这可以通过转导细胞以表达荧光蛋白来实现，无论是为了稳定表达还是作为诱导系统8，通过瞬时细胞转染，或者通过使用现在可用于活细胞标记7的细胞染料，用于活细胞跟踪以及标记亚细胞器9。
最近开发了一种用于活细胞免疫细胞化学的有用方法，其中识别所选表面标志物的抗体可以与标记试剂结合，并且在加入培养基后，表达特定标志物的细胞可以很容易地通过活细胞成像实时成像。当细胞在二维（2D）培养条件下生长时，使用这种系统可以轻松实现标记表达的可视化和定量10。
在这项研究中，我们优化了小儿弥漫性中线胶质瘤（DMG）患者来源细胞的活3D细胞免疫细胞化学侵袭和迁移的方案11,12。DMG是影响儿童的高度侵袭性脑肿瘤，绝大多数与组蛋白H3变体中的驱动突变K27M有关。DMG起源于脑干和中枢神经系统（CNS）的中线区域，其特征是高度浸润。这种侵入性能力已被证明至少部分是由肿瘤内异质性和DMG细胞的癌症干细胞样特征介导的7。
为了举例说明我们的检测方法，将抗体标记试剂（ALR）与CD44抗体结合使用。CD44是一种跨膜糖蛋白和粘附分子，在干细胞和其他细胞类型上表达，与癌症干细胞表型和肿瘤细胞迁移和侵袭有关13。这些协议包括样品制备、明场和荧光模式下的图像采集以及在活细胞分析仪器上的分析，该仪器允许在 3D 侵袭和迁移过程中实时定量测量 DMG 细胞膜上的整体 CD44 表达。这些测定还允许在迁移和入侵时可视化单个细胞上CD44的间歇荧光信号。有趣的是，还观察到抗CD44抗体的作用，其可能充当阻断抗体，似乎也减少了细胞迁移和侵袭，并诱导侵袭模式从集体间充质样转变为更单细胞的变形虫样表型。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 Well TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3595</td>
			<td>size 96 wells, polystyrene plate, flat bottom</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Euroclone</td>
			<td>ECB3056D</td>
			<td>solution for neurosphere dissociation</td>
		</tr>
		<tr>
			<td>Burker chamber</td>
			<td>Mv medical</td>
			<td>FFL16034</td>
			<td>cell counting chamber</td>
		</tr>
		<tr>
			<td>CD-44 (156-3C11)</td>
			<td>Cell Signaling Technology</td>
			<td>3570</td>
			<td>Mouse mAb IgG2a</td>
		</tr>
		<tr>
			<td>Corning Matrigel Matrix</td>
			<td>Corning</td>
			<td>356237</td>
			<td>Basement Membrane Matrix (BMM), Phenol Red-free, LDEV-free</td>
		</tr>
		<tr>
			<td>Fabfluor-488 Antibody Labeling Dye</td>
			<td>Incucyte</td>
			<td>4743</td>
			<td>Antibody labelling reagent (ALR): Mouse IgG2a 488 antibody for Live-Cell Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Incucyte S3 and/or SX5 Live-Cell Analysis Instrument</td>
			<td>Sartorius</td>
			<td>-</td>
			<td>The Incucyte S3 and/or SX5 Instrument is used for real-time cell monitoring and surveillance, cell health and viability, migration and invasion, plus a wide range of phenotypic cell-based assays.</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td></td>
			<td>-</td>
			<td>any inverted microscope</td>
		</tr>
		<tr>
			<td>Opti-Green Background Suppressor Reagent</td>
			<td>Incucyte</td>
			<td>6500-0045</td>
			<td>Backgroung suppressor reagent (BSR)</td>
		</tr>
		<tr>
			<td>Tumor stem cell (TSM) medium</td>
			<td>-</td>
			<td>-</td>
			<td>growth cell medium (see reference in the text for details)</td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment Multiple Well Plate</td>
			<td>Corning Costar</td>
			<td>7007</td>
			<td>size 96 well, round bottom clear</td>
		</tr>
	</tbody>
</table>

live-3d-cell immunocytochemistry assays of pediatric diffuse midline glioma

细胞迁移和侵袭是弥漫性中线胶质瘤（DMG）H3K27M突变肿瘤的特定标志。我们已经使用基于细胞的三维（3D）侵袭和迁移测定对这些特征进行了建模。在这项研究中，我们优化了这些用于活细胞免疫细胞化学的3D测定。抗体标记试剂用于实时检测粘附分子CD44在DMG H3K27M原代患者来源细胞系的迁移和侵袭细胞质膜上的表达。CD44与癌症干细胞表型和肿瘤细胞迁移和侵袭有关，并参与与中枢神经系统（CNS）细胞外基质的直接相互作用。在抗CD44抗体与抗体标记试剂（ALR）存在下，将来自DMG H3K27M细胞系的神经球（NS）嵌入基底膜基质（BMM）或置于BMM的薄涂层上。在活细胞分析仪器上进行活3D细胞免疫细胞化学图像分析，以定量测量整体CD44表达，特别是在迁移和入侵细胞上。该方法还允许实时可视化CD44在迁移和入侵细胞质膜上的间歇表达。此外，该测定还为CD44在DMG H3K27M细胞中间充质到变形虫转变的潜在作用提供了新的见解。

用于侵袭和迁移的活-3D细胞免疫细胞化学方案总结在 图1中简单且可重复的工作流程中。通过将DMG细胞接种在ULA 96孔圆底板中，可以获得可重现大小的NS并将其用于所示步骤。当NS达到~300μm的理想尺寸（接种后约4天）时，开始入侵12 和迁移14 测定。在单个NS的培养基中加入ALR /抗体复合物与背景抑制因子一起，允许在实时成像和随时间推移?...

Watch this Scientific Journal Video about Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma at JoVE.com

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

本研究提出了一种应用于儿科弥漫性中线胶质瘤细胞系的活 3D 细胞免疫细胞化学方案，有助于实时研究 3D 细胞侵袭和迁移等动态过程中质膜上蛋白质的表达。

小儿弥漫性中线胶质瘤的活-3D细胞免疫细胞化学测定

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

弥漫性中线胶质瘤是一种高度浸润的脑肿瘤。该方法有助于实时研究DMG细胞如何在3D环境中迁移和侵袭，并具体了解关键粘附分子的潜在作用。通过使用标记试剂和特异性抗CD44抗体，我们可以通过活细胞成像来研究CD44在DMG细胞质膜上的表达，也可以在3D迁移和侵袭中。该技术的应用突出了CD44在这些细胞运动中的潜在作用，表明它可能代表一个有价值的侵入性分子靶标。首先，通过加入 100 微升无菌水来重新水化 ALR，并通过移液混合溶液。将抗体与TSM培养基中的ALR混合在圆底多孔板或琥珀色管中，并保护其免受光照。准备足够量的培养基，以最终测定浓度的三倍每孔分配25微升，然后在室温下孵育15分钟。在TSM培养基中以1.5毫摩尔浓度稀释BSR，以在测定结束时获得0.5毫摩尔的终浓度。然后轻轻地慢慢地从每个孔中取出 75 微升培养基，避免接触 NS 所在的孔底部并目视检查 NS 的存在。向每个孔中轻轻加入 25 微升 BSR 和 ALR 抗体复合物，让 ALR 抗体复合物与培养基混合两到三分钟。使用倒置显微镜，确保每个NS位于孔底部的中心。通过使用针头去除气泡来避免气泡的形成。将盘子放在冰上五分钟，让盘子底部变冷。通过将预冷的P200尖端放在井的内壁上，每孔分配75微升BMM，避免形成气泡并接触孔底。将盘子放在冰上五分钟，让 BMM 与培养基混合。使用倒置显微镜检查NS的位置。如果不集中存在，请将板在 4 摄氏度下以 180 倍 G 离心五分钟。然后将板转移到活细胞分析仪器中，置于37摄氏度，5%二氧化碳和95%湿度的培养箱内。一旦孔涂上BMM，切割P200尖端。从每个选定的孔中取出50微升细胞培养基和NS，并将其转移到包被的平底孔中。目视检查NS在每个孔中的存在和位置。向每个孔中轻轻加入50微升稀释的BSR和ALR抗体，等待两到三分钟让试剂混合。并使用倒置显微镜，确保大多数复制的NS位于孔的中心。确保没有任何气泡后，如前所述，在活细胞分析仪器中轻轻转移板。在活细胞分析仪器软件上，选择要获取的选项计划，然后单击加号选项卡并选择按计划扫描以按文本手稿中提到的间隔扫描板。在软件窗口中，创建或恢复船只，然后单击选项新建。选择球体扫描类型、相位加明场、用于侵袭测定的绿色图像通道和4X物镜。选择稀释克隆扫描，键入4X物镜，并进行相和绿色迁移测定。选择板类型并通过在板图上突出显示要扫描的孔来定义要扫描的孔，然后设置扫描频率。单击添加到计划并开始扫描。选择“创建新分析定义”选项卡。然后在选项卡上分别选择球体侵入或基本分析仪应用程序进行入侵和迁移。在镜像通道中选择适合入侵和迁移的通道。从三到四个孔中选择一些具有代表性的图像，以预览和优化分析设置。对于侵袭测定，在分析定义选项卡中，使用文本手稿中提到的设置调整明场和绿色通道中的应用设置，以在球状体和入侵细胞之间生成精确的分割。对于迁移测定，如文本手稿中提到的相位和绿色通道调整应用设置，以在汇合细胞和绿色细胞之间生成精确的分割。通过随机单击多个孔来检查NS的分析设置是否正确。选择要分析的井和时间点。保存分析定义，然后单击完成。CD44在原发性DMG患者来源细胞中表达的代表性免疫荧光共聚焦图像证明了CD44在细胞迁移和侵袭中的作用。活体3D细胞免疫化学允许可视化CD44表达。迁移和侵袭延时的代表帧显示了随着时间的推移观察到的同一细胞上绿色荧光信号的存在和不存在，表明CD44的表达在细胞迁移和入侵时是开和关的。迁移和侵袭过程被跟踪超过96小时，显示QCTB-R059细胞具有高水平的CD44。CD44表达的定量及其随时间的增加是通过与ALR相关的迁移和侵袭的总体绿色荧光信号来测量的。当抗CD44抗体用于活细胞时，它会影响细胞形态，诱导从间充质样到变形虫样侵袭的转变，并降低这些细胞的侵袭和迁移能力。该方法最关键的步骤是将标记试剂与抗体混合，将混合物添加到基质胶和培养基中，以及访问活细胞成像仪器。该方法可以通过使用两种或两种以上独特的标记试剂和抗体来研究由直接细胞和接触介导的DMG细胞相互作用来进一步实现。我们的方法为3D研究DMG迁移和侵袭的细胞和分子机制提供了新的工具，为抑制其浸润表型提供了新的方向。

Research

JoVE Journal

Cancer Research

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

科研

癌症研究

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

将斑马鱼小儿脑肿瘤移植入免疫能力的宿主，用于长期研究肿瘤细胞行为和药物反应

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Whole-body PET/MRI of Pediatric Patients: The Details That Matter

儿科患者全身 PET/MRI: 物质的细节

Integrated PET/MRI is a hybrid imaging technique enabling clinicians to acquire diagnostic images for tumor assessment and treatment monitoring with both ...

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

石蜡嵌段细胞免疫细胞化学法测定培养体细胞蛋白质表达水平

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

石蜡包埋三维胶质瘤神经球培养的免疫组织化学评价胶质瘤生物标志物

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

多端根结核细胞迁移的实时监测

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Assays for Validating Histone Acetyltransferase Inhibitors

验证组蛋白乙酰转移酶抑制剂的检测

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

快速筛选工作流程，利用患者衍生胶质瘤干细胞确定GBM的潜在组合疗法

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...