Biology
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High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells
Chapters
Summary April 21st, 2022
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.
Transcript
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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