Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays
| Autor: | Guenther Bernhardt, Joachim Wegener, Peter Gmeiner, Christian Kade, Anne-Kathrin Mildner, Michael Skiba, Armin Buschauer, Judith A. Stolwijk, Harald Huebner |
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| Přispěvatelé: | Publica |
| Rok vydání: | 2020 |
| Předmět: |
Agonist
medicine.drug_class General Chemical Engineering Cell Endogeny Ligands General Biochemistry Genetics and Molecular Biology medicine Electric Impedance Tumor Cells Cultured Humans Receptor G protein-coupled receptor General Immunology and Microbiology Chemistry General Neuroscience Antagonist Glioma Coupling (electronics) medicine.anatomical_structure Biophysics Receptors Histamine Biological Assay Signal transduction Histamine Signal Transduction |
| Zdroj: | Journal of visualized experiments : JoVE. (156) |
| ISSN: | 1940-087X |
| Popis: | 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). |
| Databáze: | OpenAIRE |
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