Name: stepwise dosing protocol for increased throughput in label-free impedance-based gpcr assays
Uploaded: 2020-02-21T13:00:00
Description: Watch this Scientific Journal Video about Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays at JoVE.com

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 ...

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