This research was supported in part by the Intramural Research Program of the National Center for Advancing Translational Sciences, NIH. Naval Research Laboratory provided funding via its internal Nanoscience Institute. Reagent preparation was supported via the NRL COVID-19 base fund.

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
<ol>
	<li>Inside a sterile biosafety cabinet, wearing personal protective equipment (including lab gloves, lab coat, and safety glasses), prepare cell culture medium by supplementing Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), and 250 &#181;g/mL G418.
	<ol>
		<li>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.</li>
		<li>Filter through a 0.2 &#181;m filter flask and maintain sterility to avoid bacterial contamination.</li>
	</ol>
	</li>
	<li>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 &#181;L per well of cell culture medium. Fill the outer 36 wells with 100 &#181;L per well of phosphate-buffered saline (PBS).
	<ol>
		<li>To count the cells, add 2 &#181;L of acridine orange and propidium iodide stain to 18 &#181;L of cell suspension.</li>
		<li>Load 10 &#181;L of this solution into one side of a cell counting slide.</li>
		<li>Repeat steps 1.2.1. and 1.2.2. to load both sides of the slide.</li>
		<li>Place the slide into an automated cell counter.</li>
		<li>Select the fluorescence counting protocol and press Count. Count both sides of the slide and calculate the average of the two live-cell densities.</li>
		<li>To calculate the volume of cell suspension required, divide the total number of cells needed by the average cell density.</li>
	</ol>
	</li>
	<li>Inspect the wells after seeding under a light microscope to ensure that proper density and distribution have been achieved.</li>
	<li>Incubate the plate overnight in a humidified incubator at 37 &#176;C with 5% CO2.</li>
</ol>
2. Treatment of cells with QD-Spike
<ol>
	<li>Prepare 0.1% bovine serum albumin (BSA) by diluting in imaging media.
	<ol>
		<li>For 10 mL of imaging media, add 130 &#181;L of 7.5% BSA and mix.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>For 1 well of QD-Spike, add 2.27 &#181;L of 440 nM QD-Spike to 47.73 &#181;L of 0.1% BSA imaging media for a final concentration of 20 nM.</li>
		<li>To generate a six-point 1:3 serial dilution (in triplicate), add 14.26 &#181;L of QD-Spike to 285.74 &#181;L of 0.1% BSA imaging media to make the highest concentration of 20 nM. Add 100 &#181;L of 20 nM QD-Spike to 200 &#181;L 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.</li>
	</ol>
	</li>
	<li>Using a multi-channel aspirator, remove all spent media from each well. Using a multi-channel pipette, wash once with imaging media (100 &#181;L/well).</li>
	<li>Aspirate 100 &#181;L of imaging media and add back 50 &#181;L/well of QD-Spike solution.</li>
	<li>Incubate the plate for 3 h in a humidified incubator at 37 &#176;C with 5% CO2. Continue to step 4.</li>
</ol>
3. Fixation and nuclei staining
<ol>
	<li>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.</li>
	<li>Aspirate 50 &#181;L of QD-Spike from each well and add 100 &#181;L/well of 4% PFA.
	<ol>
		<li>Do not let the wells dry out. Use an automated multi-channel pipette to avoid drying of the wells (recommended).</li>
	</ol>
	</li>
	<li>Incubate for 15 min at room temperature.</li>
	<li>Wash three times with 1x phosphate-buffered saline (PBS).</li>
	<li>Prepare the deep red nuclear dye by diluting the 5 mM stock solution to 1:1000 dilution in PBS.</li>
	<li>Aspirate out the PBS and add back 50 &#181;L/well of diluted nuclear dye.</li>
	<li>Incubate for 30 min at room temperature.</li>
	<li>Wash three times with PBS.</li>
	<li>Image the plate or seal using a plate sealer for imaging at a later date. Store the plate at 4 &#176;C. Fluorescence should be stable for several weeks or more.</li>
</ol>
4. Acquisition set-up and imaging
<ol>
	<li>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.</li>
	<li>Login into the imaging platform.</li>
	<li>Create a new acquisition protocol.
	<ol>
		<li>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.</li>
		<li>Select Optical Mode as confocal and magnification as 40x water immersion.</li>
		<li>Select Binning as 1.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Select the FITC Channel for use with the ACE2-GFP cell line that allows visualization of ACE2 trafficking within the cell.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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%.</li>
		<li>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.</li>
		<li>Adjust the laser power to fine-tune the intensity of the objects of interest.</li>
	</ol>
	</li>
	<li>Save the acquisition protocol.</li>
	<li>Switch to Run Experiment and enter the Plate Name.</li>
	<li>Run the experiment.</li>
</ol>
5. Data analysis
<ol>
	<li>Import data onto the imaging software.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>In the imaging software, select Image Analysis to begin building the analysis protocol.</li>
	<li>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.</li>
	<li>Choose a well with bright cytoplasmic QD spots to begin image segmentation.</li>
	<li>In the Input Image Building Block, select Flatfield Correction.</li>
	<li>Add the next building block to the protocol by selecting Find Nuclei.
	<ol>
		<li>Here, use the DPC or far-red channel as the Nuclei marker.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Next, add the Find Cytoplasm Building Block to identify the cytoplasm.
	<ol>
		<li>In this case, use the ACE2-GFP channel for cytoplasmic segmentation.</li>
		<li>Select the method that best identifies the cytoplasm of each cell.</li>
	</ol>
	</li>
	<li>Next, add the Find Spots Building Block to identify the QD-Spike puncta.
	<ol>
		<li>Select the Nuclei as the ROI population.</li>
		<li>Select the Cell as the ROI region. This captures the puncta within the entire cell.</li>
		<li>Select the method that best identifies the QD puncta in each cell.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Add the Calculate Intensity Properties for all segmented objects (nuclei, cytoplasm/cells, and spots).</li>
	<li>Add the Calculate Morphology Properties for all segmented objects (nuclei, cytoplasm/cells, and spots).</li>
	<li>Add the Calculate Texture Properties for all segmented objects (nuclei, cytoplasm/cells, and spots).</li>
	<li>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.</li>
</ol>
6. Export data
<ol>
	<li>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.</li>
	<li>Export the dataset to a local computer or network drive.
	<ol>
		<li>Connect the image analysis software to a helper application on the computer by downloading and opening a connection file.</li>
		<li>Select the results file type (.txt, .csv, .html, or Native XML).</li>
		<li>Choose the File Folder settings with the drop-down menu.</li>
	</ol>
	</li>
</ol>
7. Analyze the data in a spreadsheet
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Plot the data in graphing software.</li>
</ol>

<ol><li>Chazotte, B. Labeling Lysosomes in Live Cells with LysoTracker. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aChazotte%2C+B+%22Cold+Spring+Harbor+Protocols%22">Cold Spring Harbor Protocols</a>. 2011 (2), 5571(2011).</li>
<li>Mehta, S., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+activity+architectures+visualized-using+genetically+encoded+fluorescent+biosensors+to+map+the+spatial+boundaries+of+signaling+compartments%5BTitle%5D)+AND+%22Accounts+of+Chemical+Research%22%5BJournal%5D)">Biochemical activity architectures visualized-using genetically encoded fluorescent biosensors to map the spatial boundaries of signaling compartments.</a> Accounts of Chemical Research. 54 (10), 2409-2420 (2021).</li>
<li>Barroso, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantum+dots+in+cell+biology%5BTitle%5D)+AND+%22The+Journal+of+Histochemistry+and+Cytochemistry%3A+Official+Journal+of+the+Histochemistry+Society%22%5BJournal%5D)">Quantum dots in cell biology.</a> The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 59 (3), 237-251 (2011).</li>
<li>Cuervo, N. Z., Grandvaux, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ACE2%3A+Evidence+of+role+as+entry+receptor+for+SARS-CoV-2+and+implications+in+comorbidities%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">ACE2: Evidence of role as entry receptor for SARS-CoV-2 and implications in comorbidities.</a> eLife. 9, 61390(2020).</li>
<li>Shang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+entry+mechanisms+of+SARS-CoV-2%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Cell entry mechanisms of SARS-CoV-2.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (21), 11727-11734 (2020).</li>
<li>Ghosh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%CE%B2-Coronaviruses+use+lysosomes+for+egress+instead+of+the+biosynthetic+secretory+pathway%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">β-Coronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway.</a> Cell. 183 (6), 1520-1535 (2020).</li>
<li>Buchser, W., et al. Assay development guidelines for image-based high content screening, high content analysis and high content imaging. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBuchser%2C+W+%22Assay+Guidance+Manual%22">Assay Guidance Manual</a>. Markossian, S., et al. , Eli Lilly & Company and the National Center for Advancing Translational Sciences. (2012).</li>
<li>Chandrasekaran, S. N., Ceulemans, H., Boyd, J. D., Carpenter, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image-based+profiling+for+drug+discovery%3A+due+for+a+machine-learning+upgrade%5BTitle%5D)+AND+%22Nature+Reviews.+Drug+Discovery%22%5BJournal%5D)">Image-based profiling for drug discovery: due for a machine-learning upgrade.</a> Nature Reviews. Drug Discovery. 20 (2), 145-159 (2021).</li>
<li>Huang, Y., Yang, C., Xu, X. F., Xu, W., Liu, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+functional+properties+of+SARS-CoV-2+spike+protein%3A+potential+antivirus+drug+development+for+COVID-19%5BTitle%5D)+AND+%22Acta+Pharmacologica+Sinica%22%5BJournal%5D)">Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19.</a> Acta Pharmacologica Sinica. 41 (9), 1141-1149 (2020).</li>
<li>Gao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SARS-CoV-2+spike+protein+interacts+with+multiple+innate+immune+receptors%5BTitle%5D)+AND+%22bioRxiv%3A+the+preprint+server+for+biology%22%5BJournal%5D)">SARS-CoV-2 spike protein interacts with multiple innate immune receptors.</a> bioRxiv: the preprint server for biology. , 227462(2020).</li>
<li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heparan+sulfate+assists+SARS-CoV-2+in+cell+entry+and+can+be+targeted+by+approved+drugs+in+vitro%5BTitle%5D)+AND+%22Cell+Discovery%22%5BJournal%5D)">Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro.</a> Cell Discovery. 6 (1), 80(2020).</li>
<li>Hoffmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SARS-CoV-2+cell+entry+depends+on+ACE2+and+TMPRSS2+and+Is+blocked+by+a+clinically+proven+protease+inhibitor%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and Is blocked by a clinically proven protease inhibitor.</a> Cell. 181 (2), 271-280 (2020).</li>
<li>Cai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+conformational+states+of+SARS-CoV-2+spike+protein%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Distinct conformational states of SARS-CoV-2 spike protein.</a> Science. 369 (6511), New York, N.Y. 1586-1592 (2020).</li>
<li>Oh, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Meta-analysis+of+cellular+toxicity+for+cadmium-containing+quantum+dots%5BTitle%5D)+AND+%22Nature+Nanotechnology%22%5BJournal%5D)">Meta-analysis of cellular toxicity for cadmium-containing quantum dots.</a> Nature Nanotechnology. 11 (5), 479-486 (2016).</li>
<li>Gorshkov, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantum+dot-conjugated+SARS-CoV-2+spike+pseudo-virions+enable+tracking+of+angiotensin+Converting+enzyme+2+binding+and+endocytosis%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Quantum dot-conjugated SARS-CoV-2 spike pseudo-virions enable tracking of angiotensin Converting enzyme 2 binding and endocytosis.</a> ACS Nano. 14 (9), 12234-12247 (2020).</li>
<li>Narayanan, S. S., Pal, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aggregated+CdS+quantum+dots%3A+Host+of+biomolecular+ligands%5BTitle%5D)+AND+%22The+Journal+of+Physical+Chemistry+B%22%5BJournal%5D)">Aggregated CdS quantum dots: Host of biomolecular ligands.</a> The Journal of Physical Chemistry B. 110 (48), 24403-24409 (2006).</li>
<li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endocytosis+of+the+receptor-binding+domain+of+SARS-CoV+spike+protein+together+with+virus+receptor+ACE2%5BTitle%5D)+AND+%22Virus+Research%22%5BJournal%5D)">Endocytosis of the receptor-binding domain of SARS-CoV spike protein together with virus receptor ACE2.</a> Virus Research. 136 (1), 8-15 (2008).</li>
<li>Hildebrandt, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Energy+transfer+with+semiconductor+quantum+dot+bioconjugates%3A+A+versatile+platform+for+biosensing%2C+energy+harvesting%2C+and+other+developing+applications%5BTitle%5D)+AND+%22Chemical+Reviews%22%5BJournal%5D)">Energy transfer with semiconductor quantum dot bioconjugates: A versatile platform for biosensing, energy harvesting, and other developing applications.</a> Chemical Reviews. 117 (2), 536(2017).</li>
</ol>

The authors have no conflicts of interest to disclose.

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...

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&#39;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 complex4,5. 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 hACE210,11. 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 shell14,15. 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.

<table><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%&lt;br/&gt; 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&#039;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 (QD&lt;sub&gt;608&lt;/sub&gt;-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

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.

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...

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

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.

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

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Biology

3.0K Views.  National Center for Advancing Translational Sciences. 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.

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

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

Immunology and Infection

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...