We thank Barbara Goricnick and Nadja Hinterreiter for their help with cell culturing and preparation of experimental solutions. The authors gratefully acknowledge financial support by the Research Training Group 1910 &#34;Medicinal chemistry of selective GPCR ligands&#34; funded by the German Research Foundation (DFG) under grant number 222125149. JAS is particularly grateful for a scholarship granted by the Bavarian Gender Equality Program.

1. Cell seeding on electrode arrays
NOTE: Selection of electrode layout is a trade-off between sensitivity and number of cells under study. The smaller the electrode, the more sensitive is the measurement but the smaller is the number of cells under study. For cells showing strong impedance fluctuations over time under baseline conditions, bigger or interdigitated electrodes are preferable.
<ol>
	<li>Pre-warm all solutions needed for standard cell passaging and seeding in a 37 &#176;C water ...

<ol>
	<li>Rosenbaum, D. M., Rasmussen, S. G., Kobilka, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+function+of+G-protein-coupled+receptors.">The structure and function of G-protein-coupled receptors.</a> Nature. 459, 356-363 (2009).</li><li>Sriram, K., Insel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+Protein-Coupled+Receptors+as+Targets+for+Approved+Drugs:+How+Many+Targets+and+How+Many+Drugs.">G Protein-Coupled Receptors as Targets for Approved Drugs: How Many Targets and How Many Drugs.</a> Molecular Pharmacology. 93, 251-258 (2018).</li><li>Kaitin, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+the+drug+development+process:+The+new+face+of+innovation.">Deconstructing the drug development process: The new face of innovation.</a> Clinical Pharmacoly and Therapeutics. 87, 6 (2010).</li><li>Kenakin, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+assays+as+portals+to+seven-transmembrane+receptor-based+drug+discovery.">Cellular assays as portals to seven-transmembrane receptor-based drug discovery.</a> Nature reviews. Drug discovery. 8, 617-626 (2009).</li><li>Zhang, R., Xie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+GPCR+drug+discovery.">Tools for GPCR drug discovery.</a> Acta Pharmacologica Sinica. 33, 372-384 (2012).</li><li>Scott, C. W., Peters, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+whole-cell+assays:+expanding+the+scope+of+GPCR+screening.">Label-free whole-cell assays: expanding the scope of GPCR screening.</a> Drug Discovery Today. 15, 704-716 (2010).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+fibroblast+behavior+in+tissue+culture+with+an+applied+electric+field.">Monitoring fibroblast behavior in tissue culture with an applied electric field.</a> Proceedings of the National Academy of Sciences of the United States of America. 81, 3761-3764 (1984).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+morphological+biosensor+for+mammalian+cells.">A morphological biosensor for mammalian cells.</a> Nature. 366, 591-592 (1993).</li><li>Fang, Y., Ferrie, A. M., Fontaine, N. H., Mauro, J., Balakrishnan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resonant+waveguide+grating+biosensor+for+living+cell+sensing.">Resonant waveguide grating biosensor for living cell sensing.</a> Biophysical Journal. 91, 16 (2006).</li><li>Stolwijk, J. A., Matrougui, K., Renken, C. W., Trebak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impedance+analysis+of+GPCR-mediated+changes+in+endothelial+barrier+function:+overview+and+fundamental+considerations+for+stable+and+reproducible+measurements.">Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.</a> Pflugers Archiv : European Journal of Physiology. 467, 2193-2218 (2015).</li><li>Lieb, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+versus+conventional+cellular+assays:+Functional+investigations+on+the+human+histamine+H1+receptor.">Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.</a> Pharmacological Research. 114, 13-26 (2016).</li><li>Stolwijk, J. A., 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=Increasing+the+throughput+of+label-free+cell+assays+to+study+the+activation+of+G-protein-coupled+receptors+by+using+a+serial+agonist+exposure+protocol.">Increasing the throughput of label-free cell assays to study the activation of G-protein-coupled receptors by using a serial agonist exposure protocol.</a> Integrative Biology. 11, 10 (2019).</li></ol>

This protocol describes a method for label-free impedance measurements to determine the dose-response relationship of agonist-induced GPCR activation in absence or presence of specific antagonists for the same receptor. The proof of concept of this method was presented in a recent publication12. To our knowledge it is the first study describing the establishment of a full dose-response curve of agonist-mediated GPCR activation using a single cell layer in vitro. The approach inevitably requires th...

This report presents a detailed description of a serial addition protocol developed for the quantification of ligand-induced G protein-coupled receptor (GPCR) activation in adherently grown cells by label-free impedance measurements. G protein-coupled receptors (GPCRs) are involved in a multitude of physiological functions and human diseases1. Because of this and their good accessibility at the cell surface, GPCRs are one of the most important drug targets. This assessment is reflected in an estimated number of ~700 approved drugs targeting GPCRs, equivalent to a ~35% share on all marketed drugs2.
The development of new drugs comprises two central processes: (i) the identification and functional characterization of biological target molecules, and (ii) the discovery of new lead substances and their development into administrable drugs. In both processes, efficient methods are required to quantitatively evaluate drug-target interactions and the subsequent biological downstream response. Different stages of the pre-clinical drug development process make use of different methods of analysis ranging from biomolecular interaction studies between drug and target, over functional studies on cells in culture, to experiments on excised organ material or whole animals. Both, physiological significance and biological complexity increase from the former to the latter3. Although it is the overall goal to minimize animal experiments, pharmacological studies using isolated organs from laboratory animals or even whole animals are regarded inevitable to comprehensively characterize new drug candidates. In terms of analytical readout, organ pharmacology studies provide a distal, integrative &#34;holistic&#34; functional response of highest physiological relevance. A drawback of such experiments is that they are not compatible with high throughput screening for technical as well as ethical reasons and have been largely substituted by studies based on in vitro cell culture4.
Methods to quantify GPCR activation in cell cultures include different label-based chemical assays, which specifically detect second messengers, phosphorylation state of downstream signaling proteins, transcriptional activation via certain transcription factors or ligand-induced intracellular receptor trafficking4,5. A drawback of such label-based assays is the necessity to label the cells with potentially harmful dyes or radioactive markers. This often requires running the assay as an endpoint-determination for an exposure time that has to be specified a priori. Using label-based endpoint assays suffers from this very limited and biased timing of the experiment and the risk that chemical labels and probes may interfere with the regular cell physiology &#8211; potentially unnoticed by the experimenter.
In recent years, label-free assays for monitoring GPCR activation have emerged, like impedance-based techniques or optical methods applying resonant waveguide gratings5,6. Labelling of the cells is experimentally not required with these approaches. As these physical readouts operate on low amplitude signals, such methods are considered non-invasive, they allow for continuous cell monitoring potentially in real time and the observation time is only limited by the cell culture not by the readout. Similar to readouts from whole organs, label-free approaches commonly report on holistic cell responses, far downstream of receptor activation when integration along the entire signaling network leads to time-dependent but rather unspecific changes in cell morphology or mass redistribution. Whereas impedance-based assays measure the dielectric signature of changes in cell shape7,8, measurements using resonant waveguide gratings are sensitive to changes in refractive index at the cell-substrate interface arising from dynamic mass redistribution (DMR)9. The integrative character makes label-free methods extremely sensitive to receptor-mediated events regardless of the type(s) of G protein (Gq, Gi/0, Gs, G12/13) or &#946;-arrestin involved in the signaling cascade6 and well-suited for endogenous expression levels of the receptor.
In a standard label-free impedance-based assay the cells are adherently grown in multi-well plates with coplanar gold-film electrodes deposited on the bottom of each well10. These electrode arrays are connected to an impedance analyzer and the cell responses to an experimental stimulus are recorded from individual wells by time-resolved impedance readings. In a typical GPCR assay a ligand is added in individually different concentrations to each individual well. The ligand-induced changes in the impedance time course are then analyzed with respect to characteristic curve features such as maximum signal change, area under the curve, signal change within a given time interval or slope of the curve at a specified time point, in order to quantify the ligand&#39;s potency and efficacy11.
The cost of the electrode arrays may limit the application of this technique in high throughput screening (HTS) campaigns. Moreover, with increasing numbers of samples to be followed in parallel, the number of individual measurements increases and thereby reduces the available time resolution for each well gradually - even for state of the art multichannel recordings. Under such conditions fast and transient cell responses may escape the measurement. In addition, the conventional one well &#8211; one concentration approach imposes a significant time and cost factor on perfused organ-on-chip or body-on-chip developments with respect to their suitability in ligand-GPCR interaction analysis.
For this reason, we developed a stepwise dosing protocol that enables recording of full dose-response curves of ligand-induced GPCR activation in cultured cell monolayers by continuously monitoring the impedance of a single well while the agonist concentration is increased stepwise. The serial agonist addition protocol significantly increases throughput per well from one concentration to 10 or more concentrations, as shown on the current example of human U-373 MG cells, which endogenously express the histamine 1 receptor (H1R). Thus, the method has the potential to significantly improve throughput in label-free dose-response studies, while the time resolution is retained at the instrumental maximum.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B&#252;rker counting chamber</td>
			<td>Marienfeld (Lauda-K&#246;nigshofen, Germany)</td>
			<td>640210</td>
			<td></td>
		</tr>
		<tr>
			<td>cell culture flasks 25 cm2</td>
			<td>Greiner bio-one (Frickenhausen, Germany)</td>
			<td>690175</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell incubator (Heraeus Function Line BB15)</td>
			<td>Thermo Scientific (Darmstadt, Germany)</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (Heraeus 1S-R)</td>
			<td>Thermo Scientific (Darmstadt, Germany)</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Diphenhydramine hydrochloride</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>D3630</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#39;s Minimum Essential Medium with 4.5 g/L D-Glucose and 2.2 g/L NaHCO3</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>Impedance Instrument (ECIS Z&#952;)</td>
			<td>Applied BioPhysics Inc. (Troy, NY, USA)</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well electrode Arrays (8W1E PET)</td>
			<td>Applied BioPhysics Inc. (Troy, NY, USA)</td>
			<td>\</td>
			<td>PET base with 0.049 mm2 working electrode and ~50 mm2 counter electrode (gold)</td>
		</tr>
		<tr>
			<td>96-well electrode arrays (96W1E+ PET)</td>
			<td>Applied BioPhysics Inc. (Troy, NY, USA)</td>
			<td>\</td>
			<td>PET base with two electrodes (gold) with 0.256 mm2 total electrode area</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Biochrom (Berlin, Germany)</td>
			<td>S0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Histamine dihydrochloride</td>
			<td>Carl Roth (Karlsruhe, Germany)</td>
			<td>4017.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood (Herasafe, KS 12)</td>
			<td>Thermo Scientific (Darmstadt, Germany)</td>
			<td>51022515</td>
			<td>class II safety cabinet</td>
		</tr>
		<tr>
			<td>Leibovitz&#39; L-15 medium</td>
			<td>Thermo Scientific (Darmstadt, Germany)</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette large (100 - 1000 &#181;L)</td>
			<td>Brandt (Wertheim, Germany)</td>
			<td>704780</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette large (20 - 200 &#181;L)</td>
			<td>Brandt (Wertheim, Germany)</td>
			<td>704778</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (phase contrast, Nikon Diaphot)</td>
			<td>Nikon (D&#252;sseldorf, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette, serological</td>
			<td>Greiner bio-one (Frickenhausen, Germany)</td>
			<td>607 180</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (accu-jet pro)</td>
			<td>Brandt (Wertheim, Germany)</td>
			<td>26300</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>T4174</td>
			<td>in PBS with 1 mM EDTA</td>
		</tr>
		<tr>
			<td>Tube, 15 mL</td>
			<td>Greiner bio-one (Frickenhausen, Germany)</td>
			<td>188 271</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, 50 mL</td>
			<td>Greiner bio-one (Frickenhausen, Germany)</td>
			<td>210 261</td>
			<td></td>
		</tr>
		<tr>
			<td>U-373 MG cells</td>
			<td>ATCC (Rockville, MD, USA)</td>
			<td>ATCC HTB-17</td>
			<td></td>
		</tr>
		<tr>
			<td>water bath (TW21)</td>
			<td>Julabo (Seelbach, Germany)</td>
			<td>\</td>
			<td></td>
		</tr>
	</tbody>
</table>

stepwise dosing protocol for increased throughput in label-free impedance-based gpcr assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

A typical scheme for preparing the various agonist solutions is shown for an experiment using 8-well electrode arrays with histamine as agonist in Tables 1-4. Table 1 and Table 2 present volumes and concentrations for an experiment using addition mode 1 (cf., Figure 1), while Table 3 and Table 4 present volumes and concentrations for an experiment following addition mode 2 (...

Watch this Scientific Journal Video about Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays at JoVE.com

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

This protocol enables the recording of a complete functional concentration response relationship of GPCR agonist from one cell population and provides detailed agonist characterization even for low cell sample numbers. The main advantage of serial dosing is the increase in throughput since a single well is sufficient for a full concentration response analysis. This technique is well suited to study single transduction in rare and complex biological models, like organ-on-chip formats in closed microfluidic containers.The protocol is not difficult to perform, provided the appropriate equipment is available. Preparing the various GPCR ligand solutions at the right concentration is the key to success. Michael Skiba, a graduate student, and Anne Mildner, an undergraduate student, from our laboratory will demonstrate the procedure.For cell seeding onto the electrode arrays, add the appropriate concentration of cells to the wells of each electrode array, and allow the cells to settle homogenously onto the well bottoms for 10 to 15 minutes at room temperature. After at least 36 hours in a standard cell culture incubator at 37 degrees Celsius and 5%carbon dioxide, inspect the cell layers by phase-contrast microscopy to ensure a complete coverage of each electrode with cells. Then replace the cell culture medium in each well with the appropriate volume of pre-warmed, serum-free medium.To start an impedance recording, place the electrode array into the connecting array holder of the impedance analyzer, and confirm a proper low impedance contact between the electrodes and the impedance analyzer. Select the electrode type and/or multi-well format in the user interface of the software. If single and multiple frequency data acquisition modes are available and the number of wells to be analyzed is low or the time resolution is not critical, select multiple frequency recordings.To ensure a maximum time resolution, select a single monitoring frequency. Then start acquisition of the time course data, and select the data folder for where to save the data. The impedance readings will be recorded at the specified number of frequencies for every well To initiate the serial addition protocol in the agonist mode for an eight-well array, add 30 microliters of the lowest concentration of the agonist of interest to the cells.Then let the cells respond and equilibrate for the predefined period of time before adding 30 microliters of the next highest concentration until each serial dilution of the agonist has been added. To analyze the data, subtract the impedance of the last data point before the first addition of agonist solution, and set the time of addition zero to normalize the impedance values. Plot the time course of normalized impedance, and plot the individual time courses to identify the maxima in impedance after each addition step.Compose a data sheet with these values, and plot the values of maximum impedance change as a function of the agonist concentration. Then use a data-fitting routine to determine the half-maximal effective concentrations and maximum response using the four-parameter logistic model. In this representative experiment, 10 solutions with increasing histamine concentrations were sequentially added to the cells every 15 minutes, and the change in impedance was measured at each time point.The impedance changes could then be plotted as a function of histamine concentration and the transfer function of a four-parameter logistic model fitted to the experimental data points from seven wells. To account for possible histamine receptor downregulation, cell desensitization, or overstimulation, the data range for the analysis can be reduced, as shown for this single serial dosing experiment. Three-parameter optimization can then be applied to the data, providing a half-maximal effective concentration of 0.75 plus or minus 0.12 micromolar for this analysis.The addition of the histamine receptor antagonist diphenhydramine 20 minutes before the first histamine results in a delayed increase of impedance within the serial dosing scheme, corresponding to the need for a higher agonist to elicit a cell response. In the dose response relationship, the effect of the antagonist is expressed as a rightward shift of the curves, corresponding to an increase in half-maximal effective concentration, provided the antagonist is a competitive ligand relative to the agonist. Successful application of the serial dosing scheme is technically easy but requires thoughtful planning, as it is challenging to perform all of the steps with the correct timing.Serial dosing has paved the way for performing concentration response studies with other readout systems that are limited with respect to the number of samples monitored in parallel. The particular advantages of serial dosing allow the study of receptor-mediated signal transduction with completely new readouts that are sensitive to other phenotypic changes, like cytomechanics. Please wear gloves and goggles when preparing the stock solutions of the receptor agonist, as they may be hazardous when inhaled or swallowed.

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6.4K vistas.  University of Regensburg. Este protocolo permite el registro de una relación de respuesta de concentración funcional completa del agonista GPCR de una población celular y proporciona una caracterización agonista detallada incluso para números de muestra de células bajos. La principal ventaja de la dosificación en serie es el aumento del rendimiento, ya que un único pozo es suficiente para un análisis completo de la respuesta de concentración. Esta técnica es adecuada para estudiar una sola transducción en ...