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 human angiotensin-converting enzyme 2 (hACE2) on the surface of human cells, subsequent internalization via fusion at the plasma membrane, and endocytosis of the Spike:hACE2 protein complex^4,5. Great insights have also been gained into the SARS-CoV-2 egress from cells via the lysosome using cellular fluorescence imaging, a unique feature of coronaviruses previously thought to occur via traditional vesicle budding from the Golgi, as it is with many other viruses⁶. A mainstay of almost all aspects of biological research, the cellular fluorescence microscopy technique has necessarily advanced in its breadth and scope of applications from super-resolution imaging of whole animals to automated high-content multi-parametric imaging for drug screening. Here, automated high-content confocal microscopy is applied to the study of SARS-CoV-2 cell entry using fluorescent QDs conjugated to the viral spike protein.

High-content analysis of images generated by biological imaging platforms allows for greater extraction of valuable biological insights than single parameters such as whole-well intensity, that one would obtain using a multi-modal plate reader⁷. By separating the objects in a field of view using automated segmentation algorithms, each object or a population of objects can be analyzed for parameters such as intensity, area, and texture in each available fluorescence channel⁸. Combining many measurements into multivariate datasets is a useful approach for phenotypic profiling. When the desired phenotype is known, such as QD internalization in the form of puncta, one can use the measurements related to puncta such as size, number, and intensity to assess the efficacy of a treatment.

Cloud-based high content imaging analysis software can accommodate a large variety of instrument data outputs, including the high content imaging platform. By using a cloud-based server for image storage and online analysis, the user is able to upload their data from either the imaging instrument or from the network drive where the data is stored. The analysis portion of the protocol is conducted within the cloud software environment, and data can be exported in a variety of file formats for downstream data visualization.

The SARS-CoV-2 virus is composed of nonstructural and structural proteins that aid in its assembly and replication. SARS-CoV-2 spike has two domains called S1 and S2, with S1 containing the receptor-binding domain responsible for hACE2 interactions at the plasma membrane⁹. Spike has also been found to interact with other molecules at the plasma membrane that may act as co-receptors in addition to hACE2^10,11. Throughout the spike protein sequence and particularly at the S1/S2 interface, there are protease cleavage sites that enable fusion at the membrane after the transmembrane serine protease 2 (TMPRSS2)¹². Various recombinant SARS-CoV-2 Spike proteins have been produced from individual receptor binding domains, to S1, S2, S1 with S2, and whole spike trimers from multiple commercial vendors for use in research activities¹³.

In this work, the surface of QDs was functionalized with recombinant spike trimers that contain a histidine tag (QD-Spike). The QDs produced by Naval Research Laboratory Optical Nanomaterials Section contain a cadmium selenide core and a zinc sulfide shell^14,15. The zinc on the QD surface coordinates the histidine residues within the recombinant protein to form a functionalized QD that resembles a SARS-CoV-2 viral particle in form and function. The generation of the nanoparticles and protein conjugation was previously described using the QD-conjugated receptor binding domain¹⁵. This method describes the cell culture preparations, QD treatment, image acquisition, and data analysis protocol that can guide a researcher in studying SARS-CoV-2 Spike activity in the physiological context of a human cell.

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 flask and maintain sterility to avoid bacterial contamination.
2. Inside a sterile biosafety cabinet, seed the interior 60 wells of a black, clear-bottom, poly-D-lysine coated 96-well plate with 20,000 cells in 100 µL per well of cell culture medium. Fill the outer 36 wells with 100 µL per well of phosphate-buffered saline (PBS).
   1. To count the cells, add 2 µL of acridine orange and propidium iodide stain to 18 µL of cell suspension.
   2. Load 10 µL of this solution into one side of a cell counting slide.
   3. Repeat steps 1.2.1. and 1.2.2. to load both sides of the slide.
   4. Place the slide into an automated cell counter.
   5. Select the fluorescence counting protocol and press Count. Count both sides of the slide and calculate the average of the two live-cell densities.
   6. To calculate the volume of cell suspension required, divide the total number of cells needed by the average cell density.
3. Inspect the wells after seeding under a light microscope to ensure that proper density and distribution have been achieved.
4. Incubate the plate overnight in a humidified incubator at 37 °C with 5% CO₂.

2. Treatment of cells with QD-Spike

1. Prepare 0.1% bovine serum albumin (BSA) by diluting in imaging media.
   1. For 10 mL of imaging media, add 130 µL of 7.5% BSA and mix.
2. In a 12-well reservoir or assay plate, dilute the 440 nM QD-Spike (SARS-CoV-2, Isolate USA-WA1/2020) stock to 20 nM using 0.1% BSA in imaging media.
   1. For 1 well of QD-Spike, add 2.27 µL of 440 nM QD-Spike to 47.73 µL of 0.1% BSA imaging media for a final concentration of 20 nM.
   2. To generate a six-point 1:3 serial dilution (in triplicate), add 14.26 µL of QD-Spike to 285.74 µL of 0.1% BSA imaging media to make the highest concentration of 20 nM. Add 100 µL of 20 nM QD-Spike to 200 µL of 0.1% BSA media to make the second dilution of 6.67 nM QD-Spike. Repeat four times to generate the six concentration points.
3. Using a multi-channel aspirator, remove all spent media from each well. Using a multi-channel pipette, wash once with imaging media (100 µL/well).
4. Aspirate 100 µL of imaging media and add back 50 µL/well of QD-Spike solution.
5. Incubate the plate for 3 h in a humidified incubator at 37 °C with 5% CO₂. Continue to step 4.

3. Fixation and nuclei staining

1. Inside a sterile biosafety cabinet, wearing personal protective equipment (including lab gloves, lab coat, and safety glasses), prepare 4% paraformaldehyde (PFA) in 0.1% BSA imaging media.
2. Aspirate 50 µL of QD-Spike from each well and add 100 µL/well of 4% PFA.
   1. Do not let the wells dry out. Use an automated multi-channel pipette to avoid drying of the wells (recommended).
3. Incubate for 15 min at room temperature.
4. Wash three times with 1x phosphate-buffered saline (PBS).
5. Prepare the deep red nuclear dye by diluting the 5 mM stock solution to 1:1000 dilution in PBS.
6. Aspirate out the PBS and add back 50 µL/well of diluted nuclear dye.
7. Incubate for 30 min at room temperature.
8. Wash three times with PBS.
9. Image the plate or seal using a plate sealer for imaging at a later date. Store the plate at 4 °C. Fluorescence should be stable for several weeks or more.

4. Acquisition set-up and imaging

1. Start the software for the imaging platform. Turn on the high content imaging platform, and the light on the status bar should be on. If the machine is not connected, the software will go into offline analysis mode.
2. Login into the imaging platform.
3. Create a new acquisition protocol.
   1. Select Plate Type as 96-well clear bottom imaging plate.
      NOTE: The plate selection in the instrument may vary and can be customized by loading the correct plate definitions that include plate dimensions such as height, width, and other distances to define well spacing and focal points.
   2. Select Optical Mode as confocal and magnification as 40x water immersion.
   3. Select Binning as 1.
   4. Choose the channels and fluorescent excitation/emission wavelengths as described in steps 4.3.5-4.3.8. Take a snapshot when the settings are selected to view the image and ensure the settings are appropriate.
      NOTE: Digital Phase Contrast (DPC) will allow visualization of live cells without a cell stain or fluorescent dye. It is not recommended for fixed cells.
   5. Select the Mode for DPC such as High Contrast to produce well-defined cell bodies. Take a snapshot to confirm that this setting is appropriate, as seen in the image window.
   6. Select the FITC Channel for use with the ACE2-GFP cell line that allows visualization of ACE2 trafficking within the cell.
   7. To make a custom channel for the QD channel, select the triangle drop-down menu for the channel and choose the excitation in the 405 nm range and emission in the 608 nm range.
   8. If cells were fixed and a deep red nuclear dye was used, select the far-red channel with emission greater than 630 nm to further delineate the cell body and act as a cell mask.
   9. Select a well with a strong cytoplasmic QD signal to set the exposure time, laser power, and Z-height position. Follow steps from 4.3.10-4.3.13.
   10. Check whether the default height produces an image where the cells are in the desired focal plane. If the endocytosed puncta are the target organelle, the Z-position will be lower than if the plasma membrane is the target organelle.
   11. Choose an exposure time that produces a bright image with gray levels at least three-fold greater than the background signal. This is typically between 100 and 300 ms. At 20 nM, the gray levels should be approximately 6000 a.u. using an exposure time of 200 ms and laser power of 80%.
   12. Check gray levels by right-clicking on the image produced with the Snapshot feature in each channel and selecting Show Intensity. Then, left-click on the object of interest or background to view the gray level for that pixel.
   13. Adjust the laser power to fine-tune the intensity of the objects of interest.
4. Save the acquisition protocol.
5. Switch to Run Experiment and enter the Plate Name.
6. Run the experiment.

5. Data analysis

1. Import data onto the imaging software.
   1. After acquiring all images, export measurements to the imaging software. This requires a server to be established and an account to be created. Create a screen for the data to be exported into the imaging software.
2. In the imaging software, select Image Analysis to begin building the analysis protocol.
3. Load the measurements from the imaging experiment by right-clicking on the file in the screens list and clicking on Select. The plate map of imaged wells and their associated fields should be visible at the bottom left-hand corner of the window.
4. Choose a well with bright cytoplasmic QD spots to begin image segmentation.
5. In the Input Image Building Block, select Flatfield Correction.
6. Add the next building block to the protocol by selecting Find Nuclei.
   1. Here, use the DPC or far-red channel as the Nuclei marker.
   2. Select the method that accurately segments the objects first, and then fine-tune the segmentation using the drop-down menu and adjusting the sliders.
      NOTE: Good segmentation accurately defines the region of interest (ROI) for the nucleus, cell, and spots. Only the pixels that are positive for the marker should be captured within an individual ROI. Background signal should be excluded. If the background is captured in the ROI, the sensitivity of the method can be decreased, or the threshold of intensity can be increased to increase the stringency of the segmentation algorithm. This is applicable to all Find building blocks.
7. Next, add the Find Cytoplasm Building Block to identify the cytoplasm.
   1. In this case, use the ACE2-GFP channel for cytoplasmic segmentation.
   2. Select the method that best identifies the cytoplasm of each cell.
8. Next, add the Find Spots Building Block to identify the QD-Spike puncta.
   1. Select the Nuclei as the ROI population.
   2. Select the Cell as the ROI region. This captures the puncta within the entire cell.
   3. Select the method that best identifies the QD puncta in each cell.
9. Once all the objects in the image have been segmented and identified accurately in this well, choose other wells to ensure the building blocks and settings can be generally applied to the other wells and other conditions.
10. Add the Calculate Intensity Properties for all segmented objects (nuclei, cytoplasm/cells, and spots).
11. Add the Calculate Morphology Properties for all segmented objects (nuclei, cytoplasm/cells, and spots).
12. Add the Calculate Texture Properties for all segmented objects (nuclei, cytoplasm/cells, and spots).
13. Define results by selecting the parameters for each population, including nuclei and spots. The number of objects can be used as an indirect measure of cell viability.

6. Export data

1. Click on Batch analysis. Wait for the analysis to finish before proceeding to the next step (step 6.2).
   NOTE: The batch analysis allows the image analysis software server to load the analysis protocol using the selected measurements to analyze the data remotely.
2. Export the dataset to a local computer or network drive.
   1. Connect the image analysis software to a helper application on the computer by downloading and opening a connection file.
   2. Select the results file type (.txt, .csv, .html, or Native XML).
   3. Choose the File Folder settings with the drop-down menu.

7. Analyze the data in a spreadsheet

1. Open the exported file. If it is a .csv file, save it as a .xls file format to enable the use of pivot tables. If the file path is too long, save the .xls file higher up in the folder hierarchy to avoid errors in saving.
2. Add columns to the spreadsheet that designate the conditions for each well. For example, the cell type, QD-Spike variants, QD-Spike concentrations, incubation time, etc.
3. Choose the Pivot Table function and build the table using the added conditions as Rows, and the measured parameters as Columns. Select the calculation for each parameter i.e., Average, Standard Deviation, Median, Min, Max, or Count.
4. Normalize the data to control samples, including unconjugated QDs as 0% and the highest concentration of QD-Spike as 100%.
   NOTE: If assessing the efficacy of inhibitors such as neutralizing antibodies, normalize the data to media-only treated cells (100% efficacy) and QD-Spike without inhibitor (0% efficacy).
5. Plot the data in graphing software.

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

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