Stepwise dosing protocol for increased throughput in label-free impedance-based GPCR assays
Stolwijk JA, Mildner AK, Kade C, Skiba M, Bernhardt G, Buschauer A, Hübner H, Gmeiner P, Wegener J (2020)
Publication Type: Journal article
Publication year: 2020
Journal
Book Volume: 2020
Article Number: e60686
Journal Issue: 156
DOI: 10.3791/60686
Abstract
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'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).
Authors with CRIS profile
Harald Hübner
Lehrstuhl für Pharmazeutische Chemie
Peter Gmeiner
Lehrstuhl für Pharmazeutische Chemie
Involved external institutions
Germany (DE)How to cite
APA:
Stolwijk, J.A., Mildner, A.-K., Kade, C., Skiba, M., Bernhardt, G., Buschauer, A.,... Wegener, J. (2020). Stepwise dosing protocol for increased throughput in label-free impedance-based GPCR assays. Journal of Visualized Experiments, 2020(156). https://dx.doi.org/10.3791/60686
MLA:
Stolwijk, Judith A., et al. "Stepwise dosing protocol for increased throughput in label-free impedance-based GPCR assays." Journal of Visualized Experiments 2020.156 (2020).
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