Classical GPCR drug screening approaches for new modulators require invasive experimental procedures that do not reveal the time course of the cell response or details about intrinsic signal amplification. We performed label-free, non-invasive impedance analysis of cells allowing monitoring the response of target cells in real time. The recording platform used was a high throughput approach enabling electrical impedance spectroscopy measurements of up to 6 × 96-well plates, simultaneously or independently. Data acquisition can be executed at 100 recording frequencies from 0.1 to 100 kHz, since the combination of data acquired at lower frequencies vs. higher frequencies provides a more comprehensive picture of cellular behavior than either measurement alone. In our study, detection of concentration-dependent signals as a result of GPCR agonist-induced morphological changes of cells was investigated, and data acquired at a cell specific, most sensitive frequency of 12 kHz. We investigated cells expressing histamine H1 receptor (H1R) or the neuropeptide-Y receptor 4 (Y4R). U373 cells endogenously expressing H1R were stimulated with agonist Histamine at 0.03–30 μM. Results show a concentration-dependent effect and the EC 50 was estimated to be approx. 690 μM. Next, 400 nM forskolin was applied to pre-stimulate CHO Y4R cells prior to the addition of the endogenous ligand, human pancreatic peptide (hPP) at concentrations ranging from 0.1–10 μM, or synthetic agonist 1 (SA1) at 0.1–1 μM. Data revealed concentration-dependent effects and the EC 50 's were estimated to be approx. 735 pM and 636 pM, respectively. In summary, the advantages of this impedance assay are (1) time resolution, (2) throughput, (3) the fact that it is label-free, and (4) high sensitivity to small but significant morphological changes that might otherwise be missed.
Wegener et al. (Sun,) studied this question.