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

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.
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregated+CdS+quantum+dots:+Host+of+biomolecular+ligands.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+of+the+receptor-binding+domain+of+SARS-CoV+spike+protein+together+with+virus+receptor+ACE2.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+transfer+with+semiconductor+quantum+dot+bioconjugates:+A+versatile+platform+for+biosensing,+energy+harvesting,+and+other+developing+applications.">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 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 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

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 ...

We have established a platform technology for SARS COVID 2 that can be applicable to other viruses with Spike mediated cell recognition and entry as the first step in cellular viral infection. Quantum dots are bright, stable, and their surface can be functionalized for a variety of cellular applications. Using this method, we can visualize and measure Spike internalization into cells.To prepare 0.1%BSA. Add 130 microliters of 7.5%BSA to 10 milliliters of imaging media in mix. For a 6.1 to three serial dilution and triplicate add 14.26 microliters of QD Spike to 285.74 microliters of the 0.1%BSA to make the highest concentration of 20 nanomole.Then add 100 microliters of 20 nanomole or QD Spike to 200 microliters of 0.1%BSA to make the second delusion of 6.67 nanomole QD Spike. Remove all spent media from each well with a multi-channel aspirator, wash once with a multi-channel pipette with 100 microliters of imaging media per well, then aspirate 100 microliters of the imaging media. Add back 50 microliters of QD Spike solution per well and incubate the plate for three hours in a humidified incubator at 37 degree Celsius with 5%carbon dioxide.Wearing protective equipment, prepare 4%PFA and 0.1%PSA imaging media inside a sterile bio safety cabinet, aspirate 50 microliters of QD Spike from each well and add 100 microliters of 4%PFA per well with an automated multichannel pipette to avoid drying the wells. Incubate for 15 minutes at room temperature, then wash three times with PBS, prepare the deep red nuclear dye by diluting the five millimeter stock solution at one to 1000 in PBS, aspirate the PBS and add back 50 microliters of diluted nuclear dye per well. Incubate for 30 minutes at room temperature then wash three times with PBS, image the plate or seal it using a plate sealer for imaging later.Store the plate at four degrees Celsius. Start the software for the imaging platform. After logging in the imaging platform create a new acquisition protocol by selecting plate type as a 96 wall transparent bottom imaging plate, select optical mode has gone focal and magnification as 40 times water immersion, select binning as one.Select the mode for digital face contrasts such as high contrast to produce well-defined cell bodies. Take a snapshot to confirm that this heading is appropriate as seen in the image window, select the fit C channel for use with the ACE2-GFP cell line to visualize ACE2 trafficking within the cell. To make a custom QD channel, select the triangle dropdown menu and choose the excitation in the 405 nanometer range in emission in the 608 nanometer range, select a well with a robust cytoplasmic QD signal to set the exposure time, laser power and Z height position.Check whether the default height produces an image of the cells in the desired focal plane. Z position will be lower for the endocytose puncta than the Z position for the plasma membrane. Choose an exposure time, typically between 100 and 300 milliseconds that produces a bright image with gray levels, at least threefold greater than the background signal.At 20 nanomole the gray levels should be approximately 6, 000 atomic units with 200 milliseconds exposure time and 80%laser power. 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, adjust the laser power to fine tune the intensity of the objects of interest. Save the acquisition protocol, switch to run experiment and enter the plate name, run the experiment.Translocation of both QDs and ACE2 was visualized using fluorescence microscopy and an ACE2-GFP expressing cell line. Image segmentation and subsequent analysis was performed to extract relevant parameters such as spot count. First, nuclei were segmented from the nuclear marker channel to create an ROI population.Then an ROI region outlining the cell with segmented using the ACE2-GFP channel. Lastly, QD 608 Spike spots were segmented from the cell ROI region, internalized QD and ACE2 signals showed strong co-localization. A concentration response experiment with six different concentrations of QD Spike was performed in HEK 293T cells.Determining optical concentrations of QD, QDS can be used as low as 2.22 nanomole, however, it is recommended to use concentrations of 10 nanomole or higher to ensure a robust response. During conjugation to the QD protein aggregation may occur. Aggregates were spotted in the QD solution as bright clumped precipitates and HEK 293T cells, QDs were incubated with neutralized antibodies starting at 30 micrograms per microliter before addition to cells, these antibodies blocked binding internalization and caused a reduction in the spot count compared to cells treated with QD only.This essay can also be used for high-throughput screening, lead evaluation of neutralizing antibodies to identify pun antivirals to the block viral entry and be adapted to study newly emerging variants.

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