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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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