Name: high-throughput confocal imaging of quantum dot-conjugated sars-cov-2 spike trimers to track binding and endocytosis in hek293t cells
Uploaded: 2022-04-21T12:30:00
Description: Watch this Scientific Journal Video about High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells at JoVE.com

这项研究得到了美国国立卫生研究院国家转化科学促进中心校内研究计划的部分支持。海军研究实验室 通过其 内部纳米科学研究所提供资金。试剂制备 通过 NRL COVID-19基础基金得到支持。

本研究中使用的HEK293T细胞系是一种永生化的细胞系。本研究未使用人类或动物受试者。
1. 细胞培养和接种
<ol><li>在无菌生物安全柜内，穿着个人防护设备（包括实验室手套、实验室外套和安全眼镜），通过用10%胎牛血清（FBS）、1%青霉素/链霉素（P/S）和250μg/mL G418补充Dulbecco的改良鹰培养基（DMEM）来制备细胞培养基。<ol>
<li>对于 500 mL 培养基，加入 443.75 mL DMEM?...

<ol>
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本文中描述的方法为使用高通量共聚焦显微镜对人细胞中的功能化量子点进行成像提供了必要的步骤。该方法最适合于内吞作用是病毒进入的主要途径而不是TMPRSS2和膜融合的活性的细胞，因为它可以研究SARS-CoV-2 Spike和hACE2内吞作用。由于QD模型的性质和市售Spike三聚体上的C端His-tag，Spike S1和S2结构域的任何TMPRSS2切割都将使QD仅附着在S2结构域12上。这可能会阻止内部化，因为 RBD ?...

荧光显微镜使研究人员能够使用专门的染料1，遗传编码的荧光蛋白2和量子点（QDs）形式的荧光纳米颗粒3来观察细胞的内部运作。对于2019年严重急性呼吸综合征冠状病毒（SARS-CoV-2）全球大流行，研究人员利用荧光显微镜来了解病毒如何在质膜和细胞质中与细胞相互作用。例如，研究人员已经能够深入了解病毒体表面的SARS-CoV-2 Spike蛋白与人类细胞表面的人血管紧张素转换酶2（hACE2）的结合，随后 通过 质膜融合的内化以及Spike：hACE2蛋白复合物的内吞作用4，5。使用细胞荧光成像对SARS-CoV-2 通过 溶酶体从细胞中流出也有很好的见解，这是冠状病毒的一个独特特征，以前被认为是 通过 高尔基体的传统囊泡萌芽发生的，就像许多其他病毒一样6。作为生物学研究几乎所有方面的支柱，细胞荧光显微镜技术在其应用的广度和范围上必然得到发展，从整个动物的超分辨率成像到用于药物筛选的自动高内涵多参数成像。在这里，自动高内涵共聚焦显微镜应用于使用与病毒刺突蛋白偶联的荧光量子点研究SARS-CoV-2细胞进入。
对生物成像平台生成的图像进行高内涵分析，可以比使用多模态读板器获得的全孔强度等单个参数更好地提取有价值的生物学见解7。通过使用自动分割算法分离视野中的物体，可以分析每个物体或一组物体，以查找每个可用荧光通道中的强度、面积和纹理等参数8。将许多测量值组合到多变量数据集中是表型分析的有用方法。当已知所需的表型时，例如Puncta形式的QD内化，可以使用与puncta相关的测量值（例如大小，数量和强度）来评估治疗的疗效。
基于云的高内涵成像分析软件可以容纳各种仪器数据输出，包括高内涵成像平台。通过使用基于云的服务器进行图像存储和在线分析，用户能够从成像仪器或存储数据的网络驱动器上传数据。该协议的分析部分在云软件环境中进行，数据可以以各种文件格式导出，用于下游数据可视化。
SARS-CoV-2病毒由非结构和结构蛋白组成，有助于其组装和复制。SARS-CoV-2尖峰具有两个称为S1和S2的结构域，其中S1包含负责质膜上hACE2相互作用的受体结合结构域9。Spike还被发现与质膜上的其他分子相互作用，这些分子除了hACE210，11之外可能还充当共受体。在整个刺突蛋白序列中，特别是在S1 / S2界面处，存在蛋白酶切割位点，可以在跨膜丝氨酸蛋白酶2（TMPRSS2）12之后的膜上融合。从单个受体结合域到S1，S2，S1和S2以及来自多个商业供应商的全穗三聚体，已经生产出各种重组SARS-CoV-2 Spike蛋白，用于研究活动13。
在这项工作中，量子点的表面被含有组氨酸标签（QD-Spike）的重组尖刺三聚体功能化。海军研究实验室光学纳米材料科生产的量子点含有硒化镉芯和硫化锌壳14，15。QD表面上的锌协调重组蛋白内的组氨酸残基，形成功能化的QD，其形式和功能类似于SARS-CoV-2病毒颗粒。纳米颗粒和蛋白质偶联的产生之前使用QD偶联受体结合域15进行了描述。该方法描述了细胞培养制剂，QD治疗，图像采集和数据分析方案，可以指导研究人员在人类细胞的生理环境中研究SARS-CoV-2 Spike活性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>32% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714</td>
			<td>Used for fixing cells after quantum dot treatment, final concentration 3.2% 
			Used for stabilizing QDs in Optimem I and preventing non-specific interactions, final concentration 0.1%</td>
		</tr>
		<tr>
			<td>7.5% Bovine Serum Albumin</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td>Used as a cell viability dye for fluorescence cell counting</td>
		</tr>
		<tr>
			<td>Acridine Orange / Propidium Iodide Stain</td>
			<td>Logos Biosystems</td>
			<td>F23001</td>
			<td>Microwell plates used for seeding cells and assaying QD-Spike</td>
		</tr>
		<tr>
			<td>Black clear bottom 96 well coated plate coated with poly-D-lysine</td>
			<td>Greiner</td>
			<td>655946</td>
			<td>Used to support cell culture, DMEM supplement</td>
		</tr>
		<tr>
			<td>Characterized Fetal Bovine Serum</td>
			<td>Cytiva/HyClone</td>
			<td>SH30071.03</td>
			<td>Cloud-based high-content image analysis software; V2.9.1</td>
		</tr>
		<tr>
			<td>Columbus Analyzer</td>
			<td>Perkin Elmer</td>
			<td>NA</td>
			<td>Used for labeling cell nuclei and cell bodies after fixation, deep red nuclear dye</td>
		</tr>
		<tr>
			<td>DRAQ5 (5 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>62252</td>
			<td>Basal media for HEK293T cell culture</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Minimal Essential Media, D-glucose (4.5g/L), L-glutamine, sodium pyruvate (110 mg/L), phenol red</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td>Used for arranging data after export from Columbus; V2110 Microsoft 365</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>NA</td>
			<td>Used to continue selection of hACE2-GFP positive cells, DMEM supplement</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>InvivoGen</td>
			<td>ant-gn-5</td>
			<td>Human embryonic kidney cell line stably expression human angiotensin converting enzyme 2 tagged with GFP</td>
		</tr>
		<tr>
			<td>HEK293T hACE2-GFP</td>
			<td>Codex Biosolutions</td>
			<td>CB-97100-203</td>
			<td>Automated cell counter</td>
		</tr>
		<tr>
			<td>Luna Automated Cell Counter</td>
			<td>Logos Biosystems</td>
			<td>NA</td>
			<td>Used for fluorescence cell counting</td>
		</tr>
		<tr>
			<td>Luna Cell Counting Slides</td>
			<td>Logos Biosystems</td>
			<td>L12001</td>
			<td>High-content imaging platform</td>
		</tr>
		<tr>
			<td>Opera Phenix</td>
			<td>Perkin Elmer</td>
			<td>NA</td>
			<td>Imaging media, used for incubating cells with quantum dots</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>11058-021</td>
			<td>Phosphate-buffered saline without calcium or magnesium used for washing cells during passaging and assaying</td>
		</tr>
		<tr>
			<td>PBS -/-</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td>Used to prevent bacterial contamination of cell culture, DMEM supplement</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Used for graphing, data visualization, and statistical analysis;V9.1.0</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td>NA</td>
			<td>Used for assaying SARS-Cov-2 Spike binding to hACE2 and monitoring Spike endocytosis</td>
		</tr>
		<tr>
			<td>Quantum Dot 608 nm-Spike (QD608-Spike)</td>
			<td>custom made by Naval Research Laboratory</td>
			<td></td>
			<td>Used for inhibition of SARS-Cov-2 Spike binding to hACE2</td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody, Mouse Mab</td>
			<td>Sino Biological</td>
			<td>40592-MM57</td>
			<td>Used to dissociate cells from flask during passaging</td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12605-010</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput confocal imaging of quantum dot-conjugated sars-cov-2 spike trimers to track binding and endocytosis in hek293t cells

细胞荧光显微镜新技术的发展促进了药物发现的高通量筛选方法。量子点是荧光纳米粒子，具有优异的光物理特性，具有明亮稳定的光致发光以及窄发射带。量子点呈球形，通过对表面化学的适当修饰，可用于共轭生物分子用于细胞应用。这些光学特性，加上用生物分子使它们功能化的能力，使它们成为研究受体 - 配体相互作用和细胞运输的绝佳工具。在这里，我们提出了一种使用量子点来跟踪SARS-CoV-2刺突蛋白的结合和内吞作用的方法。该协议可以用作实验者的指南，这些实验者希望利用量子点在细胞生理学的背景下研究蛋白质 - 蛋白质相互作用和运输。

在治疗后，量子点将被内化，因为纳米颗粒将与质膜上的ACE2结合并诱导内吞作用。使用表达ACE2-GFP的细胞系，可以使用荧光显微镜观察量子点和ACE2的易位。一旦内化，两个QD和ACE2信号显示出强烈的共定位。从这些图像中，可以执行图像分割和后续分析以提取相关参数，例如点计数（图1， 图2B）。 图1A 是用不同浓度的QD-Spike或培?...

Watch this Scientific Journal Video about High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells at JoVE.com

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

在该协议中，与重组SARS-CoV-2尖峰偶联的量子点使基于细胞的测定能够监测质膜上与hACE2的尖峰结合以及随后结合蛋白进入细胞质的内吞作用。

量子点偶联SARS-CoV-2穗状三聚体的高通量共聚焦成像，以追踪HEK293T细胞中的结合和内吞作用

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...

我们已经建立了针对SARS COVID 2的平台技术，该技术可以应用于其他病毒，Spike介导的细胞识别和进入是细胞病毒感染的第一步。量子点明亮、稳定，其表面可以针对各种蜂窝应用进行功能化。使用这种方法，我们可以可视化和测量Spike内化到细胞中。准备0.1%BSA。将130微升7.5%BSA加入10毫升混合的成像培养基中。对于6.1至3个连续稀释和一式三份，加入14.26微升QD Spike至285.74微升0.1%BSA，以达到最高浓度的20纳摩尔。然后向20.1%BSA的200微升或QD Spike中加入100微升的20.1%BSA，以产生6.67纳摩尔QD Spike的第二种错觉。用多通道吸气器从每个孔中取出所有用过的培养基，用多通道移液器洗涤一次，每孔100微升成像培养基，然后吸出100微升成像培养基。每孔回添加50微升QD Spike溶液，并将板在37摄氏度的加湿培养箱中孵育3小时，其中二氧化碳含量为5%。穿戴防护设备，在无菌生物安全柜内制备4%PFA和0.1%PSA成像培养基，从每个孔中抽出50微升QD Spike，并使用自动多通道移液器每孔添加100微升4%PFA，以避免孔干燥。在室温下孵育15分钟，然后用PBS洗涤三次，通过将5毫米的储备溶液稀释在PBS中1至1000来制备深红色核染料，吸出PBS并每孔回添加50微升稀释的核染料。在室温下孵育30分钟，然后用PBS洗涤三次，对板进行成像或使用板封口器密封，以便以后成像。将盘子存放在四摄氏度。启动映像平台的软件。在成像平台中记录后，通过选择板类型作为96壁透明底部成像板，选择光学模式已聚焦并放大为40倍水浸，选择分档作为一个。选择数字人脸对比度（如高对比度）的模式，以生成定义明确的细胞体。拍摄快照以确认此标题是否合适，如图像窗口中所示，选择适合的C通道与ACE2-GFP细胞系一起使用，以可视化细胞内的ACE2转运。要制作自定义QD通道，请选择三角形下拉菜单，然后选择405纳米范围内的激发，发射范围为608纳米，选择具有鲁棒细胞质QD信号的孔以设置曝光时间，激光功率和Z高度位置。检查默认高度是否生成所需焦平面中单元格的图像。内吞细胞点的Z位置将低于质膜的Z位置。选择曝光时间，通常在100到300毫秒之间，以产生灰度水平至少三倍的明亮图像。在20纳摩尔时，灰度水平应约为6， 000个原子单位，暴露时间为200毫秒，激光功率为80%。通过右键单击每个通道中使用快照功能生成的图像并选择显示强度来检查灰度级别，然后左键单击感兴趣的对象或背景以查看该像素的灰度级别，调整激光功率以微调感兴趣对象的强度。保存采集协议，切换到运行实验并输入板名称，运行实验。使用荧光显微镜和表达ACE2-GFP的细胞系可视化QD和ACE2的易位。执行图像分割和后续分析以提取相关参数，例如点计数。首先，将原子核从核标记通道中分离出来，以产生ROI群体。然后是一个ROI区域，使用ACE2-GFP通道勾勒出分段的像元。最后，QD 608尖峰点从小区ROI区域进行分段，内化QD和ACE2信号表现出较强的共定位。在HEK 293T细胞中进行了六种不同浓度的QD Spike的浓度反应实验。确定量子点的光学浓度，量子点可以使用低至2.22纳摩尔，但是，建议使用10纳摩尔或更高的浓度，以确保强大的响应。在与QD蛋白偶联期间可能发生聚集。在QD溶液中发现聚集物作为明亮的聚集沉淀物和HEK 293T细胞，QD与中和抗体一起孵育，从每微升30微升开始，然后加入细胞，这些抗体阻断结合内化并导致斑点计数减少与仅用QD处理的细胞相比。本文还可用于高通量筛选，中和抗体的先导评估，以识别阻断病毒进入的双关抗病毒药物，并适用于研究新出现的变异。

Research

JoVE Journal

Biology

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 ...

科研

生物学

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

在成细胞分化和融合过程中利用活成像跟踪核

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

微殖器的高通量实时成像，测量生长和基因表达中的异质性

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

用于SARS-CoV-2检测和定量的两步逆转录液滴数字PCR方案

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

城市环境 -环境水库SARS-CoV-2采样和检测管道

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

用于类器官培养和 3D 成像的高通量平台

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

Investigating Zinc Transport Mechanism in Mammalian Cells with FluoZin-3 Assay

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Exploring Cellular Zinc Regulation Through ZnT1 Functionality (Video) | JoVE

使用 体外 锌转运测定表征哺乳动物锌转运蛋白

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...