Name: Using High Content Imaging to Quantify Target Engagement in Adherent Cells (Video) | JoVE Journal
Uploaded: 2018-11-29T14:45:00.000+00:00
Duration: 7 min 23 s
Description: Learn high content imaging to quantify drug-target engagement in adherent cells using a microplate-compatible CETSA adaptation. This method enables spatial localization without cell detachment for chemical biology research.

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5 minutes

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>

<ol><li>Morgan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+a+five-dimensional+framework+on+R%26D+productivity+at+AstraZeneca%5BTitle%5D)+AND+%22Nature+Reviews+Drug+Discovery%22%5BJournal%5D)">Impact of a five-dimensional framework on R&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/pubmed?term=((The+Economics+of+Reproducibility+in+Preclinical+Research%5BTitle%5D)+AND+%22PLOS+Biology%22%5BJournal%5D)">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/pubmed?term=((An+analysis+of+the+attrition+of+drug+candidates+from+four+major+pharmaceutical+companies%5BTitle%5D)+AND+%22Nature+Reviews+Drug+Discovery%22%5BJournal%5D)">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/pubmed?term=((Can+the+flow+of+medicines+be+improved%3F+Fundamental+pharmacokinetic+and+pharmacological+principles+toward+improving+Phase+II+survival%5BTitle%5D)+AND+%22Drug+Discovery+Today%22%5BJournal%5D)">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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Target+validation+using+chemical+probes%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">Target validation using chemical probes.</a> Nature Chemical Biology. 9 (4), 195-199 (2013).</li>
<li>Schürmann, M., Janning, P., Ziegler, S., Waldmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small-Molecule+Target+Engagement+in+Cells%5BTitle%5D)+AND+%22Cell+Chemical+Biology%22%5BJournal%5D)">Small-Molecule Target Engagement in Cells.</a> Cell Chemical Biology. 23 (4), 435-441 (2016).</li>
<li>Robers, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Target+engagement+and+drug+residence+time+can+be+observed+in+living+cells+with+BRET%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Monitoring+drug+target+engagement+in+cells+and+tissues+using+the+cellular+thermal+shift+assay%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((The+Cellular+Thermal+Shift+Assay%3A+A+Novel+Biophysical+Assay+for+In+situ+Drug+Target+Engagement+and+Mechanistic+Biomarker+Studies%5BTitle%5D)+AND+%22Annual+Review+of+Pharmacology+and+Toxicology%22%5BJournal%5D)">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/pubmed?term=((The+cellular+thermal+shift+assay+for+evaluating+drug+target+interactions+in+cells%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((CETSA+screening+identifies+known+and+novel+thymidylate+synthase+inhibitors+and+slow+intracellular+activation+of+5-fluorouracil%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((In+situ+Target+Engagement+Studies+in+Adherent+Cells%5BTitle%5D)+AND+%22ACS+Chemical+Biology%22%5BJournal%5D)">In situ Target Engagement Studies in Adherent Cells.</a> ACS Chemical Biology. 13 (4), 942-950 (2018).</li>
<li>Seashore-Ludlow, B., Perspective Lundbäck, T. E. arly <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microplate+Application+of+the+Cellular+Thermal+Shift+Assay+%28CETSA%29%5BTitle%5D)+AND+%22Journal+of+Biomolecular+Screening%22%5BJournal%5D)">Microplate Application of the Cellular Thermal Shift Assay (CETSA).</a> Journal of Biomolecular Screening. 21 (10), 1019-1033 (2016).</li>
<li>Axelsson, H., Almqvist, H., Seashore-Ludlow, B., Lundback, T. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAxelsson%2C+H+%22Assay+Guidance+Manual+%5BInternet%5D%22">Assay Guidance Manual [Internet]</a>. Sittampalam, G. S., et al. , Eli Lilly & Company and the National Center for Advancing Translational Sciences. Bethesda (MD). (2016).</li>
<li>Mateus, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prediction+of+intracellular+exposure+bridges+the+gap+between+target-+and+cell-based+drug+discovery%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (30), E6231-E6239 (2017).</li>
</ol>

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

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.
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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 ...

using-high-content-imaging-to-quantify-target-engagement-adherent

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Learn high content imaging to quantify drug-target engagement in adherent cells using a microplate-compatible CETSA adaptation. This method enables spatial localization without cell detachment for chemical biology research.

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

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