The authors acknowledge infrastructure support from Science for Life Laboratory and Karolinska Institutet. The authors also acknowledge input and discussions with Michaela Vallin, Magdalena Otrocka and Thomas Lundb&#228;ck.

1. Seeding of Cells
NOTE: For a general overview of the workflow see Figure 1. A detailed list of materials and reagents are available in the Table of Materials.
<ol>
	<li>Prior to seeding of the cells, drill holes with a standard drill in the frame of black 384-well imaging assay plates to avoid air bubbles being trapped under the plate later during the heating step. To avoid plastic particles entering the wells during this step and to maintain sterile conditions, seal the plates with an adhesive aluminum foil or cover the plate prior to drilling in a tissue culture hood. Typically, 3 holes with a diameter of 3.5 mm on each of side of the plate (short edge) suffice.</li>
	<li>Prepare a laminar flow bench by cleaning with 70% ethanol. Following standard aseptic tissue culture techniques, remove media from cell flask or dish. Wash cells with 5-10 mL of phosphate-buffered saline (PBS) and then add 2 mL trypsin to the flask. Incubate the flask at 37 &#176;C until the A-431 cells detach. Count the cells either using a haemocytometer or cell counter. Prepare a cell suspension of 50,000 cells/mL in culture medium.</li>
	<li>Dispense 40 &#181;L of cell suspension (giving a final cell density of 2,000 cells per well) into each well of an assay plate using a bulk reagent dispenser or a multichannel pipet, depending on the scale of the experiment. Briefly move the plate from side-to-side to disperse cells evenly on the bottom of the plate.</li>
	<li>To minimize plate-edge effects, allow the cells to settle at the bottom of the assay plate for 20 minutes at room temperature in the back of the laminar flow hood. Then, place the plate in a plastic container with damp paper towels to ensure a humid atmosphere. Prior to use, wipe the plastic container with 70% ethanol.</li>
	<li>Incubate the box with the assay plate for 2-3 days at 37 &#176;C and 5% CO2 in a conventional humidified incubator. Monitor confluency of the cells until 50-75%, as assessed by visual inspection with a light microscope.</li>
</ol>
2. Compound Treatment
<ol>
	<li>On the day of the experiment, aspirate the medium from each well using a plate washer. Place the plate on the plate washer and select the aspiration program. If a plate washer is not available, then the liquid can be removed by inverting the plate with a rapid hand twist over a waste tray or sink. Complete removal of liquid is essential for good performance. Any excess liquid is then removed by dabbing with paper towels.</li>
	<li>Add 30 &#181;L of compounds diluted to the appropriated concentration in cell culture medium using automated dispensing or a multichannel pipette depending on the scale of the experiment. Ensure to add a negative (DMSO) and positive (known ligand) control to several wells on each assay plate. Since this is a thermal shift assay, it is necessary that the compound concentration exceeds the dissociation constant to observe protein stabilization. Thus, a rough guideline for compound concentration is 50-100 times the IC50, but more detail descriptions for compound concentrations are found in the Discussion section. 
	NOTE: DMSO tolerability of the cell lines should be tested prior to the experiment.</li>
	<li>Seal the compound-treated assay plate with a breathable plate seal and incubate at 37 &#176;C and 5% CO2 in a humidified incubator for 30 minutes.</li>
</ol>
3. Heat Challenge
<ol>
	<li>First, set the water bath to the desired temperature. Note that the final temperature that is reached inside the wells of the assay plate can be different from the final temperature in the water bath. Investigate the offset beforehand with a dummy plate and thermocouple thermometer. It typically takes 30 minutes for the bath to stabilize at the desired temperature.</li>
	<li>To verify that the desired temperature is reached in the wells of the assay plate during the heating step, prepare an unsealed dummy plate containing the same volume of medium as the assay plate.</li>
	<li>Remove the assay plates from the incubator. Take off the breathable seal and re-seal the assay plate containing the compound-treated cells with a tight adhesive aluminum foil to ensure that no water will leak into the wells during the subsequent heating in the water bath. Ensure that the drilled holes in plate frame are accessible.</li>
	<li>Place the assay plate and the dummy plate in the water bath with the bottom of the plate angled towards the water surface to force any remaining air out from under the plates.</li>
	<li>Monitor the temperature inside the wells of a new dummy plate using a thermocouple thermometer.</li>
	<li>Heat the assay plate in the water bath for 3 minutes. Immediately transfer the assay and dummy plate to another water bath with room-tempered water to cool down for 5 minutes. The assay plate is now ready for further processing.</li>
</ol>
4. Fixation
<ol>
	<li>Dispense 10 &#181;L 16% (w/v) paraformaldehyde (PFA) directly to the assay plate using a bulk reagent dispenser or a multichannel pipet. Incubate at room temperature for 20 minutes. 
	Note: Some fixatives are classified as carcinogenic and institutional safety regulations should be followed.</li>
	<li>Aspirate the PFA solution and wash the cells with 300 &#181;L PBS using a plate washer. Place the plate on the plate washer and select the aspiration program. 
	NOTE: This procedure has been optimized using an overflow protocol on the plate washer in which liquid is simultaneously dispensed and removed. If a plate washer or similar procedure is not available, the washing step can alternatively be done manually.</li>
	<li>Manual washing procedure: Remove the liquid from the wells by inverting the plate with a rapid hand twist over a waste tray or sink. Complete removal of liquid is essential for good performance. Add 80 &#181;L of PBS with a multichannel pipette and invert the plate again as described above, repeat the washing procedure two times. After the last wash, blot the plate against clean paper towels to remove any excess liquid.</li>
</ol>
5. Permeabilization
<ol>
	<li>Add 20 &#181;L of 0.1% (v/v) NP-40 to the wells with a multichannel pipet and incubate at room temperature for 10 minutes. Wash the cells using the same procedure as described above (step 4.2).</li>
	<li>Alternatively, when appropriate, apply an antigen retrieval protocol, e.g.:
	<ol>
		<li>Add 20 &#181;L of 1% SDS to the wells with a multichannel pipet. Incubate at room temperature for 5 minutes and wash according to the same procedure described above (step 4.2).</li>
		<li>Add 80 &#956;L of 10 mM glycine at pH 7.2 and incubate for 10 minutes at room temperature. Aspirate the glycine solution using a plate washer by placing on the plate washer and selecting the aspiration protocol. See notes in 2.1 and 4.2 if a plate washer is not available.</li>
	</ol>
	</li>
</ol>
6. Blocking
<ol>
	<li>Add 15 &#181;L of 1% (w/v) bovine serum albumin (BSA) in PBS to the wells using a bulk reagent dispenser or a multichannel pipet. Incubate the plate at room temperature for 1 hour or overnight at 4 &#176;C with an aluminum foil plate seal.</li>
</ol>
7. Primary Antibody
<ol>
	<li>Aspirate the blocking solution using a plate washer by placing on the plate washer and selecting the aspiration protocol. See notes in 2.1 and 4.2 if a plate washer is not available.</li>
	<li>Add 10 &#181;L of primary antibody diluted accordingly in 1% (w/v) BSA in PBS to the wells using a multichannel pipet. Incubate the plate at room temperature for 1 hour or overnight at 4 &#176;C with an aluminum foil plate seal.</li>
</ol>
8. Secondary Antibody
<ol>
	<li>Aspirate the primary antibody solution and wash the wells according to same procedure as described above (step 4.2).</li>
	<li>Add 10 &#181;L of Alexa 488 secondary antibody diluted accordingly in 1% (w/v) BSA in PBS. Incubate at room temperature for 1 hour. Seal the plate with an adhesive aluminum foil seal to protect from light. 
	NOTE: Protect the plate from light during this and subsequent steps.</li>
</ol>
9. Nuclear Staining and Cell Mask
<ol>
	<li>Add 10 &#181;L of nuclear dye diluted to 0.05 mg/mL in PBS to the wells using a multichannel pipet. Incubate at room temperature for additional 10 minutes.</li>
	<li>Aspirate the secondary antibody and Hoechst solution and wash the wells using the same procedure as described above (step 4.2).</li>
	<li>Add 10 &#181;L of cell mask diluted to 200 ng/mL in PBS to the wells using a multichannel pipet. Incubate at room temperature for 30 minutes.</li>
	<li>Aspirate the cell mask solution and wash the wells using the same procedure as described above (step 4.2).</li>
	<li>Dispense 60 &#181;L of PBS to all wells using the plate washer, a bulk reagent dispenser, or a multichannel pipet, and seal the plates with an adhesive aluminum foil.</li>
</ol>
10. Image Acquisition and Analysis
<ol>
	<li>Capture images on a high content imager using 3 fluorescent channels: DAPI (387/447), GFP (472/520), and TexasRed (562/624). Acquire 4 images per well using 10X objective. Use automated laser autofocus and apply binning 2 during acquisition. Store images as 16 bit, gray scale tiff files along with metadata.</li>
	<li>Analyze images using available software. Identify cell boundaries using a Cell Scoring algorithm with DAPI (nucleus) and TexasRed (cytoplasm).</li>
	<li>Extract average intensity for all acquired wavelengths for further data analysis.</li>
	<li>Calculate the Z-factor to ensure the robustness of the assay.</li>
	<li>Calculate % stabilization using the following formula: 100*(1-(well intensity &#8211; average well intensity negative control)/(average intensity positive control-average intensity negative control)). Here negative control is DMSO and positive control is the reference substance. Since the maximum stabilization between compounds can vary and in fact be greater than the positive control for ITDRF curves, the maximum and minimum stabilization intensities for each compound are sometimes used in place of the intensity values of the control wells.</li>
</ol>

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	<li>Morgan, P., 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=Impact+of+a+five-dimensional+framework+on+R&amp;D+productivity+at+AstraZeneca.">Impact of a five-dimensional framework on R&amp;D productivity at AstraZeneca.</a> Nature Reviews Drug Discovery. 17 (3), 167-181 (2018).</li><li>Freedman, L. P., Cockburn, I. M., Simcoe, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Economics+of+Reproducibility+in+Preclinical+Research.">The Economics of Reproducibility in Preclinical Research.</a> PLOS Biology. 13 (6), e1002165 (2015).</li><li>Waring, M. J., 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=An+analysis+of+the+attrition+of+drug+candidates+from+four+major+pharmaceutical+companies.">An analysis of the attrition of drug candidates from four major pharmaceutical companies.</a> Nature Reviews Drug Discovery. 14 (7), 475-486 (2015).</li><li>Morgan, P., 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=Can+the+flow+of+medicines+be+improved?+Fundamental+pharmacokinetic+and+pharmacological+principles+toward+improving+Phase+II+survival.">Can the flow of medicines be improved? Fundamental pharmacokinetic and pharmacological principles toward improving Phase II survival.</a> Drug Discovery Today. 17 (9), 419-424 (2012).</li><li>Bunnage, M. E., Chekler, E. L. P., Jones, L. 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B., 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=Target+engagement+and+drug+residence+time+can+be+observed+in+living+cells+with+BRET.">Target engagement and drug residence time can be observed in living cells with BRET.</a> Nature Communications. 6, 10091 (2015).</li><li>Martinez Molina, D., 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=Monitoring+drug+target+engagement+in+cells+and+tissues+using+the+cellular+thermal+shift+assay.">Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay.</a> Science. 341 (6141), 84-87 (2013).</li><li>Martinez Molina, D., Nordlund, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cellular+Thermal+Shift+Assay:+A+Novel+Biophysical+Assay+for+In+situ+Drug+Target+Engagement+and+Mechanistic+Biomarker+Studies.">The Cellular Thermal Shift Assay: A Novel Biophysical Assay for In situ Drug Target Engagement and Mechanistic Biomarker Studies.</a> Annual Review of Pharmacology and Toxicology. 56, 141-161 (2016).</li><li>Jafari, R., 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=The+cellular+thermal+shift+assay+for+evaluating+drug+target+interactions+in+cells.">The cellular thermal shift assay for evaluating drug target interactions in cells.</a> Nature Protocols. 9 (9), 2100-2122 (2014).</li><li>Almqvist, H., 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=CETSA+screening+identifies+known+and+novel+thymidylate+synthase+inhibitors+and+slow+intracellular+activation+of+5-fluorouracil.">CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil.</a> Nature Communications. 7, 11040 (2016).</li><li>Axelsson, H., 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=In+situ+Target+Engagement+Studies+in+Adherent+Cells.">In situ Target Engagement Studies in Adherent Cells.</a> ACS Chemical Biology. 13 (4), 942-950 (2018).</li><li>Seashore-Ludlow, B., Perspective Lundb&#228;ck, T. 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The authors have no disclosures to report.

As discussed in the results section, there are several key steps to the procedure. First, it is important to identify a high-quality affinity reagent. We recommend screening a small library of antibodies for each desired target. After a primary antibody has been selected, it is also important to validate the system for a number of different binding sites of the protein target if appropriate. Counter-screening for compounds that interfere with the assay signal as shown in Figure 2C by omittin...

When developing new drugs or chemical probes it is essential to couple the observed pharmacological effect or functional readout to measurements of target occupancy or engagement in live cells1,2,3. These data are necessary both to ensure that the small molecule in fact reaches its desired target and to validate the biological hypothesis behind protein target selection4,5. Furthermore, during drug development, model systems of increasing complexity are used to select and corroborate a lead compound prior to clinical trials. To confirm translation of biology across these preclinical systems, methods for tracing drug-target engagement and accompanying biology throughout this development process are critical.
Drug-target engagement has traditionally been challenging to monitor in live cells with unfunctionalized small molecules and proteins, especially at the single-cell level with spatial resolution6,7. One recent method to observe the interaction between unmodified drugs and proteins in live cells is the cellular thermal shift assay (CETSA) in which ligand-induced stabilization of a native protein in response to a heat challenge is quantified8,9,10. This is accomplished by quantifying remaining soluble protein after exposure to a heat challenge. In the initial disclosure of CETSA, western blot was used for detection. To enable screening campaigns and hit triaging of larger compound collections, efforts to increase the throughput of CETSA experiments have lead to the development of several homogenous, microplate-based assays10,11. However, one limitation with these methods is that they are currently best suited to compound treatment in cell suspensions and the detection requires cell lysis, leading to loss of spatial information. CETSA can be applied experimentally either as a ligand-induced shift in thermal aggregation temperature (Tagg) at a single concentration of the small molecule or the ligand concentration necessary to stabilize the protein at a single temperature. The latter is termed isothermal dose response fingerprints (ITDRF) to signify the dependence of these measurements on the specific experimental conditions.
The goal of this protocol is to measure target engagement using CETSA in adherent cells by immunofluorescent (IF) antibody detection with high-content microscopy12. This procedure extends the original CETSA platform to allow for single-cell quantification of target engagement with conservation of subcellular localization. Notably, unlike many previous reports, in this procedure compound treatment is performed in live adherent cells without surface detachment or washing prior to the heat challenge, thus preserving the established binding equilibrium we aim to measure13. Currently, the method is validated for one target protein p38&#945; (MAPK14) in several cell lines, and we hope that by sharing this procedure the technique can be applied broadly across the melting proteome. We anticipate that this protocol can be adapted throughout the drug development pipeline from screening, hit triaging through to monitoring of target engagement in vivo.

<table><tbody><tr><td>Phosphate-buffered saline (PBS)</td><td>Medicago</td><td>09-9400-100</td><td></td></tr><tr><td>TrypLE Express</td><td>ThermoFisher Scientific</td><td>12604013</td><td>for detaching cells and subculturing</td></tr><tr><td>16% paraformaldehyde (PFA)</td><td>ThermoFisher Scientific</td><td>28908</td><td>fixative</td></tr><tr><td>Goat anti-rabbit IgG (H+L), Alexa Fluor 488 conjugated antibody</td><td>ThermoFisher Scientific</td><td>A11008</td><td>secondary antibody</td></tr><tr><td>HCS CellMask Red stain</td><td>ThermoFisher Scientific</td><td>H32712</td><td>Cytoplasm stain</td></tr><tr><td>NP-40</td><td>Sigma-Aldrich</td><td>56741</td><td>for permeabilization</td></tr><tr><td>Hoechst stain 33342</td><td>Sigma-Aldrich</td><td>B2261</td><td>nuclear stain</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM) - high Glucose</td><td>Sigma-Aldrich</td><td>6429</td><td>cell culture media component</td></tr><tr><td>Heat-inactivated fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F9665</td><td>cell culture media component</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td>cell culture media component</td></tr><tr><td>Corning, breathable plate seal</td><td>Sigma-Aldrich</td><td>CLS3345</td><td>for copound incubation step</td></tr><tr><td>Rabbit anti-p38 antibody [E229]</td><td>Abcam</td><td>ab170099</td><td>primary antibody, LOT:GR305364-16</td></tr><tr><td>Falcon, Black 384-well clear bottom imaging plates</td><td>VWR</td><td>736-2044</td><td>imaging plates</td></tr><tr><td>Greiner, 384-well low volume polypropylene plates</td><td>VWR</td><td>784201</td><td></td></tr><tr><td>Adhesive aluminum foil</td><td>VWR</td><td>30127790</td><td></td></tr><tr><td>Peelable aluminium seal</td><td>Agilent</td><td>24210-001</td><td>for PlateLoc</td></tr><tr><td>LY2228820</td><td>Selleckchem</td><td>S1494</td><td>p38&amp;alpha; inhibitor</td></tr><tr><td>PH797804</td><td>Selleckchem</td><td>PH797804</td><td>p38&amp;alpha; inhibitor</td></tr><tr><td>BIRB796</td><td>Selleckchem</td><td>S1574</td><td>p38&amp;alpha; inhibitor</td></tr><tr><td>SB203580</td><td>Tocris</td><td>1202</td><td>p38&amp;alpha; inhibitor</td></tr><tr><td>AMG 548</td><td>Tocris</td><td>3920</td><td>p38&amp;alpha; inhibitor</td></tr><tr><td>RWJ 67657</td><td>Tocris</td><td>2999</td><td>p38&amp;alpha; inhibitor</td></tr><tr><td>L-Skepinone</td><td>CBCS compound collection</td><td></td><td>p38&amp;alpha; inhibitor</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A7030</td><td>blocking agent</td></tr><tr><td>SDS (sodium dodecyl sulfate)</td><td>BDH</td><td>44244</td><td>used in antigen retrieval</td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8898</td><td>used in antigen retrieval</td></tr><tr><td>A-431 cells</td><td>ATCC</td><td>ATC-CRL-1555</td><td></td></tr><tr><td>Echo 550</td><td>Labcyte</td><td></td><td>For preparation of compound plates</td></tr><tr><td>Plate sealer</td><td>Agilent</td><td></td><td>PlateLoc</td></tr><tr><td>Bulk reagent dispenser</td><td>Thermo Scientific</td><td>5840300</td><td>Multidrop Combi</td></tr><tr><td>Automated liquid handling</td><td>Agilent</td><td></td><td>Bravo liquid handling platform; used for compound plate preparation</td></tr><tr><td>Plate washer</td><td>Tecan</td><td></td><td>Hydrospeed</td></tr><tr><td>Water bath</td><td>Julabo</td><td>TW12</td><td></td></tr><tr><td>Thermocouple</td><td>VWR</td><td></td><td>Thermocouple traceable lab thermometer</td></tr><tr><td>High content imager</td><td>Molecular Devices</td><td></td><td>ImageXpress Micro XLS Widefield High-Content Analysis System</td></tr></tbody></table>

using high content imaging to quantify target engagement in adherent cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

The protocol outlined in Figure 1 describes the basic workflow for running CETSA assays on adherent cells with detection of remaining soluble protein by high content imaging. This workflow can be easily adapted to all stages of assay development by modifying the plate layout of the compounds or reagents14. We detail expected results for several anticipated use cases below.
<e...

Watch this Scientific Journal Video about Using High Content Imaging to Quantify Target Engagement in Adherent Cells at JoVE.com

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

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Research

JoVE Journal

Biochemistry

8.5K Views.  Karolinska Institutet, Solna. Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

Video: Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Immunology and Infection

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

Cancer Research

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Developmental Biology

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Bioengineering

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Biology

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate multiple functions, including adhesion, proliferation, migration, and differentiation. Mechanical forces can be transmitted from the cell via the adhesion receptor integrin to ligands in the ECM. The amount and spatial organization of these cell-generated forces can be modulated by growth factor receptors, including epidermal growth factor receptor (EGFR). The tools currently available to quantify crosstalk-mediated changes in cell mechanics and relate them to&#160;focal adhesions, cellular morphology, and signaling are limited. DNA-based molecular force sensors known as tension gauge tethers (TGTs) have been employed to quantify these changes. TGT probes are unique in their ability to both modulate the underlying force threshold and report piconewton scale receptor forces across the entire adherent cell surface at diffraction-limited spatial resolution. The TGT probes used here rely on the irreversible dissociation of a DNA duplex by receptor-ligand forces that generate a fluorescent signal. This allows quantification of the cumulative integrin tension (force history) of the cell. This article describes a protocol employing TGTs to study the impact of EGFR on integrin mechanics and adhesion formation. The assembly of&#160;the TGT mechanical sensing platform is systematically detailed and the procedure to image forces, focal adhesions, and cell spreading is outlined. Overall, the ability to modulate the underlying force threshold of the probe, the adhesion ligand, and the type and concentration of growth factor employed for stimulation make this a robust platform for studying the interplay of diverse membrane receptors in regulating integrin-mediated forces.

TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell spreading.

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

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Real-time Live Imaging of T-cell Signaling Complex Formation

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

Quantitation of Endothelial Cell Adhesiveness In Vitro

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion