Name: simultaneous imaging and flow-cytometry-based detection of multiple fluorescent senescence markers in therapy-induced senescent cancer cells
Uploaded: 2022-07-12T14:30:00
Description: Watch this Scientific Journal Video about Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells at JoVE.com

这项工作得到了约翰内斯·开普勒大学林茨分校（BERM16108001）对Yu勇的资助。

1. DLBCL细胞系与马磷酰胺或柔红霉素治疗，诱导细胞衰老
注意：该协议也适用于贴壁癌细胞。根据细胞大小，将1-2×105 个细胞放入6孔板的一个孔中，并将板在5%CO2，37°C培养箱中孵育过夜，然后在处理前。方案步骤与悬浮细胞相同，但有两个例外。首先，需要在步骤3.4之后将细胞从板上胰蛋白酶消化。其次，在胰蛋白酶消化之前无需离心即可执行洗...

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该方法通过明场成像和流式细胞术定量检查了化疗后四种不同DLBCL细胞系的衰老进入能力。在单细胞水平上，我们成功地在经过处理的KARPAS422和WSU-DLCL2细胞中检测到主要的C12FDG + EdU-Ki67 +衰老群体，并且在较小程度上在OCI-LY1细胞中检测到，而SU-DHL6细胞系对治疗具有抗性。细胞系之间衰老进入能力的差异可以通过其独特的基因组缺陷来解释16，</...

多种刺激可引发细胞衰老，使细胞进入细胞周期停滞的稳定状态。这些刺激包括内在信号变化或外在应力。内在信号包括进行性端粒缩短、端粒结构改变、表观遗传修饰、蛋白稳态障碍、线粒体功能障碍和癌基因激活。外在应激包括炎症和/或组织损伤信号、辐射或化学治疗以及营养剥夺1，2，3，4。在不同类型的衰老中，最常见和研究最充分的是复制性衰老、癌基因诱导的衰老 （OIS）、辐射诱导的衰老和治疗诱导的衰老 （TIS）。OIS是对异常癌基因激活产生的复制应激引起的遗传毒性损伤的急性细胞反应，并且可以在一定程度上阻止从肿瘤前病变到完全肿瘤的病理进展。当肿瘤细胞受到化疗药物或电离辐射5，6的压力时，就会发生TIS。
衰老被认为是病理学中的双刃剑，因为它具有高度的动态性质。它最初被描述为一种有益的肿瘤抑制机制，从分裂细胞的循环池中去除受损细胞，保护器官的正常功能并抑制肿瘤生长7，8，9。然而，新出现的证据表明衰老有黑暗的一面。衰老细胞分泌促炎细胞因子，称为衰老相关分泌表型（SASP），导致纤维化和器官功能障碍，并促进肿瘤的发生和进展10。此外，衰老的癌细胞与染色质重塑和持续DNA损伤反应（DDR）的激活同时进行表观遗传和基因表达重编程11，12，新获得新的癌症干细胞特性3。虽然与衰老能力较弱的肿瘤相比，衰老功能的肿瘤对治疗干预的反应更好13，但如果衰老细胞不能被高效识别和消除，则衰老细胞的持续存在可能导致长期预后不良5。无论哪种方式，评估衰老的可靠方法都具有重要的临床意义，不仅对于治疗的预后，而且对于开发针对衰老细胞的新策略。
无论触发因素如何，衰老细胞都表现出一些共同的特征，包括增大、扁平、多核形态与大液泡、细胞核显著扩张、细胞核中形成富含H3K9me3的衰老相关异染色质（SAHF）、DNA损伤标记物γH2AX病灶的持续积累、活化的p53-p21CIP1和Rb-p16INK4a 细胞周期调节机制、稳定的 G1 细胞周期停滞、SASP 的大量诱导以及衰老相关的β半乳糖苷酶 （SA-β-gal） 活性升高14.由于没有单一标记物足以定义衰老，因此SA-β-gal活性的酶染色被认为是衰老检测的金标准，通常与H3K9me3和Ki67的免疫组化染色相结合以检测TIS15。然而，基于化学显色的SA-β-gal很难量化。在这里，我们将5-十二酰氨基荧光素-二-β-D-半乳糖基糖苷（C12FDG）荧光基SA-β-gal（fSA-β-gal）检测与γH2AX和EdU掺入的DNA的免疫荧光染色相结合，使用先进的成像流式细胞仪系统鉴定C12FDG + EdU-γH2AX +衰老细胞，该系统将速度，灵敏度和详细的单细胞图像与流式细胞术和显微镜无法提供的空间信息相结合。该方法可以快速生成高分辨率图像，从而允许对细胞内的荧光信号进行定位和定量，同时通过构建标准管道获得对多个样品的快速分析的许可。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 647 anti-H2A.X Phospho (Ser139) Antibody</td>
			<td>Biolegend</td>
			<td>613407</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ki-67 Mouse Monoclonal Antibody (Alexa Fluor 647)</td>
			<td>Biolegend</td>
			<td>350509</td>
			<td></td>
		</tr>
		<tr>
			<td>C12FDG (5-Dodecanoylaminofluorescein Di-&#946;-D-Galactopyranoside)</td>
			<td>Fisher Scientific</td>
			<td>11590276</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroquin -diphosphat</td>
			<td>Sigma aldrich</td>
			<td>C6628</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleanser (Coulter Clenz)</td>
			<td>Beckman Coulter</td>
			<td>8546929</td>
			<td></td>
		</tr>
		<tr>
			<td>Click-iT EdU Pacific Blue Flow Cytometry Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>C10418</td>
			<td></td>
		</tr>
		<tr>
			<td>Daunorubicin</td>
			<td>Medchemexpress</td>
			<td>HY-13062A</td>
			<td></td>
		</tr>
		<tr>
			<td>Debubbler (70% Isopropanol)</td>
			<td>Millipore</td>
			<td>1.3704</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis software (Amnis&#160;IDEAS&#160;6.3)</td>
			<td>Luminex</td>
			<td>CN-SW69-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument and imaging software (Amnis&#160;ImageStreamX&#160;Mk II Imaging Flow Cytometer System and INSPIRE software)</td>
			<td>Luminex</td>
			<td>100220</td>
			<td></td>
		</tr>
		<tr>
			<td>KARPAS</td>
			<td>DSMZ</td>
			<td>ACC 31</td>
			<td></td>
		</tr>
		<tr>
			<td>mafosfamide cyclohexylamine</td>
			<td>Niomech</td>
			<td>D-17272</td>
			<td></td>
		</tr>
		<tr>
			<td>OCI-LY1</td>
			<td>DSMZ</td>
			<td>ACC 722</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>11473704</td>
			<td></td>
		</tr>
		<tr>
			<td>PETG (2-Phenylethyl-&#946;-D-thiogalactosid)&#160;</td>
			<td>Sigma aldrich</td>
			<td>P4902</td>
			<td></td>
		</tr>
		<tr>
			<td>saponin</td>
			<td>Sigma aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheath</td>
			<td>Millipore</td>
			<td>BSS-1006-B</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedBead Kit for ImageStream</td>
			<td>Luminex</td>
			<td>400041</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizer (0.4-0.7% Hypochlorite)</td>
			<td>VWR</td>
			<td>JT9416-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-DHL6</td>
			<td>DSMZ</td>
			<td>ACC 572</td>
			<td></td>
		</tr>
		<tr>
			<td>WSU-DLCL2</td>
			<td>DSMZ</td>
			<td>ACC 575</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous imaging and flow-cytometry-based detection of multiple fluorescent senescence markers in therapy-induced senescent cancer cells

化疗药物可诱导癌细胞中不可修复的DNA损伤，导致细胞凋亡或过早衰老。与凋亡细胞死亡不同，衰老是抑制癌细胞繁殖的根本不同机制。几十年的科学研究揭示了衰老癌细胞在肿瘤和调节癌细胞和基质细胞的微环境中的复杂病理作用。新的证据表明，衰老是癌症治疗过程中的一个有效预后因素，因此快速准确地检测癌症样本中的衰老细胞至关重要。本文提出了一种可视化和检测癌细胞中治疗诱导的衰老（TIS）的方法。弥漫性大B细胞淋巴瘤（DLBCL）细胞系用马磷酰胺（MAF）或柔红霉素（DN）处理，并检查衰老标志物，衰老相关β-半乳糖苷酶（SA-β-gal），DNA合成标志物5-乙炔基-2′-脱氧尿苷（EdU）和DNA损伤标志物γ-H2AX（γH2AX）。流式细胞仪成像可以帮助在短时间内生成高分辨率的单细胞图像，以同时可视化和量化癌细胞中的三种标志物。

使用图像分析软件通过加载单色控制样品的记录数据来生成补偿矩阵。如 补充图S1所示，检测到从EdU到C 12 FDG的不可忽略（系数值≥0.1）光溢出，串扰系数值为0.248，而其他通道之间的串扰不显着。用5μg/ mL MAF或20 ng / mL DN处理四种不同的DLBCL细胞系以诱导细胞衰老，并使用常规SA-β-gal染色或成像流式细胞术方法进行分析。
超过70%的KARPAS422，WSU-DLCL2和OCI-LY1?...

Watch this Scientific Journal Video about Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells at JoVE.com

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

在这里，我们提出了一种基于流式细胞术的方法，用于可视化和定量单个细胞中多种衰老相关标记物。

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

该方法可以快速生成高分辨率图像，允许分析细胞内荧光信号的空间分布和定量，同时允许快速分析多个样品。使用先进的成像流式细胞术系统测量细胞，将速度、灵敏度和详细的单细胞图像与空间信息相结合，产生流式视光学或显微镜无法提供的独特数据。一个重要的建议是提供足够的电池来建立仪器设置，我们将细胞以高密度悬浮以进行测量。除此之外，数据采集非常简单，类似于传统的流式细胞术。首先生成手稿中描述的DLBCL细胞系。然后将10毫摩尔EdU溶液以1：1000的比例加入DLBCL细胞培养物中，并将板在37摄氏度的5%二氧化碳培养箱中孵育3小时，然后通过轻轻摇动混合板。孵育后取出板，加入100毫摩尔氯喹溶液至终浓度为75微摩尔。轻轻摇动混合并孵育 30 分钟。然后加入20毫摩尔C12 FDG溶液至终浓度为20微摩尔。轻轻混合后，如前所述将板孵育一小时。接下来，以 1 至 50 的比例加入 100 毫摩尔 2-苯乙基-β-d-硫代半乳糖苷溶液以停止 FSA β-半乳糖染色。并轻轻旋转盘子进行混合。将细胞转移到15毫升无菌离心管中，并在4摄氏度下以100倍G旋转5分钟。丢弃上清液后，用四毫升PBS洗涤细胞，并在4摄氏度下以100倍G离心5分钟。然后将细胞沉淀在4%多聚甲醛固定溶液中的500微升中重新悬浮。在室温下孵育10分钟后，在室温下以250倍G离心5分钟。弃去上清液，用四毫升PBS洗涤细胞两次，并在室温下以100倍G离心5分钟。洗涤后，弃去上清液并将细胞沉淀重新悬浮在200微升皂苷透化缓冲液中。现在将悬浮液转移到新的1.5毫升管中，并在室温下孵育10分钟。在室温下以250倍G沉淀细胞五分钟。然后将细胞重新悬浮在200微升一抗溶液中，并在黑暗中以4摄氏度孵育过夜。在 250 倍 G 下以 4 摄氏度离心管五分钟。弃去上清液，用100微升皂苷洗涤液洗涤。洗涤细胞两次后，弃去上清液并将细胞沉淀重新悬浮在500微升EdU检测混合物中。在室温下在黑暗中孵育30分钟，并在室温下以250倍G离心5分钟。弃去上清液，用一毫升皂苷洗涤液洗涤。重复洗涤两次，弃去上清液并将细胞沉淀重新悬浮在20至50微升PBS中。在打开仪器之前，清空废液瓶并检查速珠、灭菌器、清洁器、除泡器和护套的液位是否充足。打开仪器和成像软件后，单击启动按钮以初始化流体和系统校准。将放大倍率设置为 40 倍，并将流体速度设置为低。打开激光器后，打开405纳米激光测量EdU Pacific Blue，488纳米测量C12-FDG，642纳米测量Alexa-Fluor-647-γ-H2AX。为散射通道设置通道六，为明场设置通道 1 和通道 9。然后从预期具有最高荧光的样品开始，以设置激光的强度，并轻轻翻转样品管进行混合。打开试管盖后，将样品管插入底座上。单击加载以开始并打开散点图。接下来，选择 特征 区域，下划线 MO1 和纵横比下划线 MO1 分别用于 X 轴和 Y 轴。将上方的门设置为0.5，以排除下方和右侧的双峰和细胞聚集体以及左侧的SpeedBead群。现在，打开直方图并为X轴选择掩码1和通道1的特征梯度均方根。选择单重态群体并设置门以选择聚焦细胞。打开直方图并为 X 轴通道 2、7 和 11 选择原始最大像素强度。然后分别调整通道二、七和 11 的 488 纳米、405 纳米和 642 纳米激光器的激光功率，使每个荧光染料的原始最大像素值在 100 到 4， 000 之间，以避免过饱和。选择要记录的重点人群，然后单击“获取”以一致的设置测量DLBCL样本。更换样品进行测量时，单击返回以恢复样品管。按加载以丢弃样本。测量完所有样品后，关闭明场并散射激光。测量单色对照样品以生成补偿矩阵。单击关机关闭成像系统。在图像分析软件中分析数据。使用图像分析软件的点向导工具自动计数和量化核伽马 H2AX 病灶和活细胞图像。选择两个细胞群，一个具有高斑点计数，一个具有低斑点计数，以训练斑点向导以进行进一步的自动斑点计数分析。单细胞图像中的流式细胞术方法显示C12-FDG阳性，EdU阴性，γH2AX阳性，衰老群体和KARPAS422增加。WSU-DLCL2和OCI-LY1细胞，但不在SU-DHL6细胞中。基于成像的分析显示γ H2AX病灶和KARPAS422，WSU-DLCL2和OCI-LY1的数量显着增加，但在SU-DHL6细胞中则不然。频率和定量数据表示了类似的结果。在细胞悬液中添加皂苷至关重要，否则抗体无法到达细胞内部抗原，刺激性去垢剂会导致C12-FTG信号丢失 成像细胞术是分析标志物定位变化的最佳选择，例如转录因子对某些刺激的易位到细胞核。这样的分析也可以通过我们的协议来实现。该方法允许在单细胞水平上可视化多个荧光标记物，这有助于检测单个信号的存在、强度和定位。

Research

JoVE Journal

Cancer Research

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

流动 Cytometry-based 药物筛选系统对促进细胞分化胶质母细胞瘤小分子的鉴定

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

科研

癌症研究

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Detection of Protease Activity by Fluorescent Peptide Zymography

荧光肽酶谱法检测蛋白酶活性

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

超声成像对小鼠肺肿瘤进展的检测

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

斑马鱼中具有功能化荧光硅纳米粒子的异种移植人类癌细胞的Vivo靶向

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Far-Red Fluorescent Senescence-Associated ÃƒÅ½Ã‚Â²-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

远红荧光衰老相关β-半乳糖苷酶探针，用于通过流式细胞术鉴定和富集衰老肿瘤细胞

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

通过颈内动脉注射癌细胞模拟脑转移

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

一种基于流式细胞术的细胞表面蛋白结合测定，用于评估抗癌适配体的选择性和特异性

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...