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Stepwise dosing protocol for increased throughput in label-free impedance-based GPCR assays

: Stolwijk, J.A.; Mildner, A.-K.; Kade, C.; Skiba, M.; Bernhardt, G.; Buschauer, A.; Huebner, H.; Gmeiner, P.; Wegener, J.


Journal of visualized experiments : JoVE. Online resource (2020), No.156, Art. e60686
ISSN: 1940-087X
Journal Article
Fraunhofer EMFT ()

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 GP CR 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).