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

摘要

In this protocol, quantum dots conjugated to recombinant SARS-CoV-2 spike enable cell-based assays to monitor spike binding to hACE2 at the plasma membrane and subsequent endocytosis of the bound proteins into the cytoplasm.

摘要

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

引言

Fluorescence microscopy enables researchers to peer into the inner workings of the cell using specialized dyes¹, genetically encoded fluorescent proteins², and fluorescent nanoparticles in the form of quantum dots (QDs)³. For the severe acute respiratory syndrome coronavirus of 2019 (SARS-CoV-2) global pandemic, researchers have employed fluorescence microscopy to understand how the virus interacts with the cell both at the plasma membrane and in the cytoplasm. For example, researchers have been able to gain insights into the binding of the SARS-CoV-2 Spike protein on the virion's surface to h....

方案

The HEK293T cell line used in this study is an immortalized cell line. No human or animal subjects were used in this study.

1. Cell culturing and seeding

1. Inside a sterile biosafety cabinet, wearing personal protective equipment (including lab gloves, lab coat, and safety glasses), prepare cell culture medium by supplementing Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), and 250 µg/mL G418.
   1. For 500 mL of media, add 443.75 mL of DMEM, 50 mL of FBS, 5 mL of P/S, and 1.25 mL of G418.
   2. Filter through a 0.2 µm filter fla....

结果

Upon treatment, the QDs will be internalized as the nanoparticle will bind to ACE2 on the plasma membrane and induce endocytosis. Using an ACE2-GFP expressing cell line, translocation of both QDs and ACE2 can be visualized using fluorescence microscopy. Once internalized, the two QD and ACE2 signals show strong colocalization. From these images, image segmentation and subsequent analysis can be performed to extract relevant parameters such as spot count (Figure 1, Figure.......

讨论

The method described in this article provides the necessary steps for imaging functionalized QDs in human cells using high-throughput confocal microscopy. This method is best suited for cells where endocytosis is the main route of viral entry rather than the activity of TMPRSS2 and membrane fusion, as it enables the study of SARS-CoV-2 Spike and hACE2 endocytosis. Because of the nature of the QD model and the C-terminal His-tag on the commercially available Spike trimer, any TMPRSS2 cleavage of Spike S1 and S2 domains wo.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
32% 多聚甲醛	Electron Microscopy Sciences	15714	用于量子点处理后的细胞固定，终浓度 3.2% 用于稳定 Optimem I 中的 QD 并防止非特异性相互作用，终浓度 0.1%
7.5% 牛血清白蛋白	Gibco	15260-037	用作细胞活力染料，用于荧光细胞计数
吖啶橙/碘化丙啶染料	Logos Biosystems	F23001	微孔板，用于接种细胞和检测 QD-Spike
黑色透明底 96 孔涂层板，包被有聚-D-赖氨酸	Greiner	655946	用于支持细胞培养、DMEM 补充剂
特征胎牛血清	Cytiva/HyClone	SH30071.03	基于云的高内涵图像分析软件;V2.9.1
哥伦布分析仪	Perkin Elmer	NA	用于标记固定后的细胞核和细胞体，深红色核染料
DRAQ5 （5 mM）	ThermoFisher Scientific	62252	用于HEK293T细胞培养的基础培养基
Dulbecco 最低必需培养基，D-葡萄糖 （4.5g/L），L-谷氨酰胺，丙酮酸钠 （110 mg/L），酚红	Gibco	11995-065	用于排列从 Columbus 导出后的数据;V2110 Microsoft 365
Excel	Microsoft	NA	用于继续选择 hACE2-GFP 阳性细胞，DMEM 补充剂
G418	InvivoGen	ant-gn-5	人胚胎肾细胞系稳定表达用 GFP 标记的人血管紧张素转换酶 2
HEK293T hACE2-GFP	Codex Biosolutions	CB-97100-203	自动细胞计数仪
Luna 自动细胞计数仪	Logos Biosystems	NA	用于荧光细胞计数
Luna 细胞计数载玻片	Logos Biosystems	L12001	高内涵成像平台
Opera Phenix	Perkin Elmer	NA	成像培养基，用于用量子点孵育细胞
Opti-MEM I 减血清培养基	Gibco	11058-021	不含钙或镁的磷酸盐缓冲盐水，用于传代和检测过程中洗涤细胞
PBS -/-	Gibco	10010-023	用于防止细胞培养物、DMEM 补充剂
青霉素 链霉素	Gibco	15140-122	用于绘图、数据可视化和统计分析;V9.1.0
Prism	GraphPad	NA	用于检测 SARS-Cov-2 刺突与 hACE2 的结合并监测刺突内吞作用
点 608 nm-Spike （QD608-Spike）	海军研究实验室		用于抑制 SARS-Cov-2 刺突与 hACE2 的结合
SARS-CoV-2 （2019-nCoV） 刺突中和抗体，小鼠单克隆	义翘神州	40592-MM57	用于在传代过程中从培养瓶中解离
TrypLE Express	Gibco	12605-010