Name: extracellular vesicle uptake assay via confocal microscope imaging analysis
Uploaded: 2022-02-14T13:00:00
Description: Watch this Scientific Journal Video about Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis at JoVE.com

这项工作得到了NCI拨款号的支持。U54CA143803、CA163124、CA093900 和 CA143055 至 K. J. P.这项研究得到了韩国健康产业发展研究所（KHIDI）韩国健康技术研发项目的资助，该研究由韩国卫生福利部资助（批准号：HI19C1122）。J. Kim和Y.-K的作品Cho得到了韩国政府资助的基础科学研究所（IBS-R020-D1）的支持。作者感谢布雷迪泌尿外科研究所的现任和前任成员，特别是Pienta-Amend实验室的成员，对手稿的批判性阅读。

1. EV分离和片上免疫荧光EV标记
<ol><li>细胞培养基（CCM）的收集和CCM的预处理，用于EV分离<ol>
<li>将PC3细胞以30%汇合度接种在75 cm 2 细胞培养瓶中。允许对照细胞在标准培养基和细胞系特异性补充剂中生长至90%汇合度（约48小时）。 注意：为了防止含EV的成分影响细胞摄取（即胎儿牛血清），请使用外泌体耗尽的培养基和补充剂。</li>
		<li>收获CCM。</li>
		<li>在室温（RT）下以1000× g ...

<ol>
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通过共聚焦显微镜进行基于3D荧光成像的EV摄取测定提供了一种有效的方法和灵敏的分析。这种荧光EV标记有助于EV的可视化，并成功执行精确的EV摄取测定。以前用于标记EV和去除残留染料的方法已经通过使用超速离心（UC）去除沉淀来报道;然而，UC可能会共同沉淀EV，并且固定化的染料可能导致假阳性信号12，13。基于纳米过滤的微流体装置消除了...

细胞外囊泡（EV）是纳米大小的脂质膜结合颗粒，按其大小分类：外体（100-500nm）和外泌体（50-150nm）1。EV含有各种生物分子，如蛋白质、核酸和脂质。这些生物分子起源于细胞，然后被封装为货物并通过EV1，2，3释放到细胞外空间。
由于其货物的多样性，电动汽车被认为在细胞间通信中发挥着积极作用。细胞释放和摄取EV允许生物分子在细胞之间转移4，5。将EV货物引入细胞可能会改变受体细胞的功能和稳态4，5，6。电动汽车通过多种途径内化;然而，确切的机制尚未得到准确证明。
大多数EV摄取测定，如遗传标记，荧光标记单个EV7。由此产生的信号可以通过微孔板光度计，流式细胞术或显微镜进行测量，每种技术都有很大的局限性。微孔板光度计、流式细胞术或标准二维（2D）显微镜无法区分内化和浅表附着的EV8，9。此外，这些技术中的每一种的必要样品制备都可能给EV摄取评估带来其他问题。例如，在EV摄取分析之前用胰蛋白酶提升粘附的细胞可能会切割细胞表面的一些表面附着的EV10，11。胰蛋白酶也可能与细胞表面相互作用，影响细胞和EV表型。此外，胰蛋白酶可能不会完全分离浅表的EV，从而扭曲孤立的人群。
为了用荧光染料准确标记EV，需要额外的洗涤步骤来去除残留的染料7。公认的隔离技术也可能导致由于EV隔离期间发生的凝血而导致假阳性信号。例如，连续超速离心（UC）被广泛用于分离EV和去除固定化染料。然而，UC可能会共同沉淀EV，并且残留的染料可能导致假阳性信号12，13。其他纳滤方法，如基于色谱柱的过滤，也广泛用于非固定化染料去除。EV和染料在柱基质内相互作用的复杂性质可能导致残留染料的不完全去除，因为复合物输入改变了色谱柱的分子截止14，15，16。
目前的方案提出了一种基于纳米过滤的微流体装置，用于分离和洗涤荧光标记的分离EV。基于纳滤的微流体装置 可以通过 流体辅助分离技术（FAST）17，18提供有效的过滤。FAST降低了过滤器的压降，从而减少了电动汽车和染料之间的潜在聚集。通过有效去除残留染料，可以提高荧光标记EV的质量和测定的特异性。
共聚焦显微镜可以区分细胞表面内化和浅表附着的EV，并在时空分辨率下全面研究EV摄取的细胞机制19，20，21，22，23，24，25。例如，Sung等人使用他们开发的活细胞报告器描述了外泌体生命周期的可视化。使用三维（3D）和后图像处理工具中的共聚焦显微镜检测和分析内化EV的位置20。虽然小型EV（40-200nm）的尺寸低于光学显微镜的分辨率极限，但由于光电探测器可以检测到增强的荧光发射，因此可以通过共聚焦显微镜检测荧光标记的EV。因此，通过获取EV和周围细胞器的多个z堆叠图像，可以精确地确定细胞内荧光标记的EV的亚细胞定位。
此外，3D重建和后数据处理可以进一步了解内部化，浅表和自由浮动的电动汽车的定位。通过将这些过程与共聚焦显微镜提供的延时活细胞成像相结合，可以精确评估EV摄取的水平，并且还可以实时跟踪EV摄取。此外，通过评估EV与细胞器的共定位，可以使用共聚焦显微镜进行EV贩运分析，这是确定内化EV如何参与细胞内功能的第一步。该协议描述了使用基于纳滤的微流体装置17，26，共聚焦显微镜和图像后分析进行EV摄取测定的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 anti-human CD63 Antibody</td>
			<td>Biolegend</td>
			<td>353038</td>
			<td>Fluorescent dye conjugated EV-specific antibody</td>
		</tr>
		<tr>
			<td>CellTracker Orange CMTMR Dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C2927</td>
			<td>Live cell (cytoplasm) fluoresent labeling reagent</td>
		</tr>
		<tr>
			<td>CFI Apo Lambda S 40XC WI</td>
			<td>Nikon</td>
			<td>MRD77400</td>
			<td>Objective for confocal imaging, NA=1.25</td>
		</tr>
		<tr>
			<td>CFI Plan Apo VC 20X</td>
			<td>Nikon</td>
			<td>MRD70200</td>
			<td>Objective for confocal imaging, NA=0.75</td>
		</tr>
		<tr>
			<td>Exodisc</td>
			<td>Labspinner Inc.</td>
			<td>EX-D1001</td>
			<td>A nano-filtration based microfluidic device for EV isolation</td>
		</tr>
		<tr>
			<td>ExoDiscovery</td>
			<td>Labspinner Inc.</td>
			<td>EX-R1001</td>
			<td>Operation device for Exodisc</td>
		</tr>
		<tr>
			<td>Exosome-depleted FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2720801</td>
			<td>Nutrient of cell culture media for PC3 cell line derived EV collection</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>VWR</td>
			<td>1500-500</td>
			<td>Nutrient for cell cultivation</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG H&#38;L preadsorbed</td>
			<td>abcam</td>
			<td>&#160;ab7063</td>
			<td>Mouse IgG antibody for negatvie control of EV labeling</td>
		</tr>
		<tr>
			<td>Ibidi USA&#160;U DISH &#956;-Dish 35 mm</td>
			<td>Ibidi</td>
			<td>81156</td>
			<td>Culture dish for confocal imaging</td>
		</tr>
		<tr>
			<td>Imaris 9.7.1</td>
			<td>Oxford Instruments</td>
			<td>9.7.1</td>
			<td>Post-image processing software</td>
		</tr>
		<tr>
			<td>Incubator System+ CO2/O2/N2 gas mixer</td>
			<td>Live Cell Instrument</td>
			<td>TU-O-20</td>
			<td>Incubator system for live cell imaging</td>
		</tr>
		<tr>
			<td>Nikon A1 HD25 / A1R HD25 camera</td>
			<td>Nikon</td>
			<td>NA</td>
			<td>Camera for confocal imaging</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti microscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td>Inverted microscope for confocal imaging</td>
		</tr>
		<tr>
			<td>NIS-Elements AR 4.50.00</td>
			<td>Nikon</td>
			<td>4.50.00</td>
			<td>Image processing software for Nikon microscope</td>
		</tr>
		<tr>
			<td>NTA, NanoSight NS500</td>
			<td>Malvern Panalytical</td>
			<td>NS500</td>
			<td>Measurement device for EV concentration</td>
		</tr>
		<tr>
			<td>OriginPro 2020</td>
			<td>OriginLab</td>
			<td>9.7.0.185</td>
			<td>Graphing software</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Antibiotics for cell cultivation</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>21875034</td>
			<td>Cell culture media for PC3 cell line cultivation</td>
		</tr>
		<tr>
			<td>SYTO RNASelect Green Fluorescent cell Stain - 5 mM Solution in DMSO</td>
			<td>Thermo Fisher Scientific</td>
			<td>S32703</td>
			<td>RNA staining fluorescent dye for the EV labeling</td>
		</tr>
	</tbody>
</table>

extracellular vesicle uptake assay via confocal microscope imaging analysis

需要实际的测定来可视化和量化细胞的细胞外囊泡（EV）摄取。EV摄取在各个研究领域的细胞间通讯中发挥作用;癌症生物学，神经科学和药物输送。文献中已经报道了许多EV摄取测定;然而，缺乏实用，详细的实验方法。可以通过荧光标记EV来评估EV的摄取，以检测它们在细胞中的位置。区分细胞内化的EV和细胞上的浅表EV对于准确确定EV摄取是困难的，但至关重要。因此，在这项工作中提出了一种通过三维（3D）荧光共聚焦显微镜有效量化EV摄取的测定方法。使用基于纳滤的微流体装置制备荧光标记的EV，通过3D共聚焦显微镜可视化，然后通过先进的图像处理软件进行分析。该协议为在细胞水平上分析电动汽车提供了一种强大的方法，并为有效分析提供了一种实用的方法。

使用基于纳米过滤的微流体装置，从PC3 CCM中分离EV并用荧光团偶联的EV特异性（CD63）抗体标记（图1）。标记的EV通过3D共聚焦显微镜成功可视化（图2）。标记的EV与细胞在外泌体耗尽的培养基中孵育数小时。孵育后，用外泌体耗尽的培养基洗涤细胞。其余的EV在孵育期间被内化或粘附在细胞上。已标记单元格区域。内化的EV被可视化为puncta19<sup class="xref"...

Watch this Scientific Journal Video about Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis at JoVE.com

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

细胞外囊泡（EV）有助于细胞生物学和细胞间通讯。需要实际的测定来可视化和量化细胞对EV的摄取。目前的方案提出了EV摄取测定，通过共聚焦显微镜利用三维荧 光成像， 然后通过基于纳滤的微流体装置进行EV分离。

通过共聚焦显微镜成像分析进行细胞外囊泡摄取测定

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...

该协议为在细胞水平上分析EV提供了一种强大的方法，并为有效的EV摄取和EV跟踪分析提供了一种实用的方法。这种EV标记方法消除了EV和染料的共沉淀，从而增强了EV信号。通过体积分析来测量EV的吸收，以区分内化的EV和浅表的EV。电动汽车是活跃的货物，因此，它们的吸收允许生化分子的转移。对于基于EV的药物递送和癌症治疗的研究，该技术可以成为评估工具。EV摄取在细胞间通讯中发挥作用，并在各种研究领域进行研究，例如癌症生物学，神经科学和药物递送。该协议可能有助于有效的EV摄取测定。为了分离细胞外囊泡或EV，将一毫升预处理的细胞培养基注入基于纳滤的微流体装置的样品室中。要操作设备，请在台式纺纱机中以 3000 RPM 旋转设备 10 分钟。纺丝后，通过移液或吸气从废物室中除去液体，然后重复此过程两次以处理另外两毫升的细胞培养基。接下来，要清洗隔离的EV，将一毫升PBS注入样品室，然后在台式纺纱机中旋转设备。对于EV的免疫荧光标记，将每毫升EV特异性抗体注射一微克到含有100微升分离EV的装置的洗脱孔中。然后在黑暗中在平板振荡器上孵育一小时，以确保抗体在样品上的均匀分布。接下来，将胶带粘附在洗脱孔上。将一毫升PBS注入样品室以洗出残留抗体，然后旋转设备直到样品室空无一人。通过将另一毫升PBS注入样品室并旋转设备（如前所述）来重复洗涤。从腔室中除去残留液体后，将膜室中荧光标记的EV移液到琥珀色管或微型管中。在使用前保护管子免受光照。用一毫升培养基将4次10至第四个PC3细胞接种到35毫升培养皿中。让细胞在最佳细胞培养条件下粘附过夜。第二天，用外泌体耗尽的培养基洗涤粘附的细胞两次。使用外泌体耗尽的培养基将荧光标记的EV稀释至适当的浓度后，将稀释的EV加入粘附的靶细胞中并孵育所需的实验时间。通过用无外泌体培养基洗涤细胞三次去除非内化EV后，用每毫升CMTMR一微克标记粘附细胞的细胞质，并在最佳细胞培养条件下孵育。对于活细胞成像，将制备的细胞置于舞台培养箱中。根据对照样品设置成像参数后，确定目标细胞的深度和Z方向上的堆叠大小范围以获取3D共聚焦图像，然后将图像采集设置为同时具有细胞特异性染料和EV特异性染料的多个Z堆叠图像。对于图像处理，请使用自动图像处理软件。要构建单元格的虚拟曲面，请单击"添加新曲面"。要配置虚拟像元表面，请单击添加按钮，然后选择质量作为过滤器类型。通过目视检查阈值下限的适当值，设置上限的最大值，然后单击"完成"按钮 接下来，要构建EV的虚拟点，请单击按钮添加新点。在"算法设置"下，选择"不同光斑大小"和"最短距离计算"，然后单击"下一步"并选择通道 1 Alexa Fluor 488 作为源通道。在"点检测"下输入估计 XY 直径的适当值，然后单击下一步。要配置虚拟 EV 点，请单击添加按钮，然后选择质量作为过滤器类型。通过目视检查设置阈值下限，然后单击下一步 对于点区域类型，选择绝对强度，然后单击下一步。要对 EV 点的区域设置阈值，请为"通过目视检查的区域阈值"输入适当的值。在"区域体积"下选择"直径"，然后单击"完成"。要拆分构建曲面内的分组点，请单击构建点并进入过滤器。接下来，单击"添加"按钮，然后选择"到曲面的最短距离"、"曲面曲面 1"作为过滤器类型。为下限设置最低阈值并为上限设置适当的值后，单击"将截面复制到新的 Spots"按钮。要自动计数单元格内的 EV，请单击"已建点 1"选项，转至"统计信息"，然后从"点总数"中导出值。要获取像元数，请单击"构建曲面 1"，然后转至统计数据并导出"总体曲面总数"的值。最后，转到"统计信息"下的"详细信息"选项卡以导出卷。使用基于纳滤的微流体装置从PC3细胞培养基中分离的EV用氟-4偶联EV特异性抗体标记，并使用3D共聚焦显微镜可视化。内化的EV被可视化为puncta，并且对这些图像的单独 EV.Post 处理允许将EV内化可视化和量化到细胞中。EV摄取测定结果表明，EV摄取水平取决于潜伏期的长度。内化EV的数量可以归一化为受体细胞体积，以确定指定细胞的实际EV摄取速率。该程序允许系统地排除非内部化电动汽车，以精确测量内部化电动汽车的数量。此处显示了内化 EV 点的大小分布。因此，关键步骤是利用微流体装置进行EV标记，在可视化荧光EV时最小化背景荧光，然后进行成像后分析以确定内化与浅表的EV。因此，可以在3D平面上实时执行细胞内EV跟踪，此外，还可以实现空间/时间分辨率的EV贩运分析以及EV与亚细胞器的共定位。因此，我们相信这种技术为电动汽车细胞通信领域的研究人员提供了一种更简单，更有效的方法来研究细胞内和细胞外基质中的EV。

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Biology

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Unit 1

Endocytosis and Exocytosis

SCIENTISTS IN ACTION

Proper Care and Cleaning of the Microscope

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

科研

生物学

Major Components of the Light Microscope

光镜下的主要组成部分

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

基于细胞健康监测的实时细胞成像分析对低密度脂蛋白胆固醇的吸收检测

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

通过纳流式细胞术表征单个细胞外囊泡跨膜蛋白

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

使用体外和体内模型分析细胞外囊泡介导的血管钙化

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; ...

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

多路复用表面等离子体共振成像芯片上的多模态分析平台，用于分析细胞外囊泡亚群

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Advancing Mitochondrial Research Â– mtHyper7 Biosensor for Subcellular Analysis (Video) | JoVE

mtHyPer7（一种用于线粒体过氧化物的比例生物传感器）在活酵母细胞中的成像

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...

Intravital Imaging Using an Inverted Microscope to Track Skin Cell Dynamics

Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope

Developing a Tool for Using Inverted Confocal Microscopes for In Vivo Intravital Imaging (Video) | JoVE

简化活体成像方法，用于在倒置共聚焦显微镜上长期监测上皮组织动力学

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It ...