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>

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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 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 ...

high-throughput-confocal-imaging-quantum-dot-conjugated-sars-cov-2

Research

JoVE Journal

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

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

Investigating Zinc Transport Mechanism in Mammalian Cells with FluoZin-3 Assay

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Author Spotlight: Exploring Cellular Zinc Regulation Through ZnT1 Functionality 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...