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
Book Volume: 2020
Article Number: e60686
Journal Issue: 156
DOI: 10.3791/60686
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).
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://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).
BibTeX: Download