title: Label-free analysis of GPCR-stimulation: The critical impact of cell adhesion.
abstract: Label-free cell-based assays have been attracting growing attention in drug research. Optical approaches based on evanescent electric fields (e.g. EPIC, RWG/DMR, SPR) and electrochemical impedance analysis (ECIS, xCELLigence) are by far the most widespread techniques for such purposes. We compared three label-free approaches (ECIS, RWG/DMR and SPR) with respect to the activation of the human histamine H1receptor (H1R) expressed by U-373 MG glioblastoma and genetically engineered HEK 293T cells. HEK 293T cells were either expressing the hH1R alone or in combination with the adhesion protein hMSR1. The β2-adrenergic receptor (β2-AR) expressed by bovine aortic endothelial cells (BAEC) served as a second cell model. Reduced cell adhesion to the surface of the sensing devices affected both, the optical and the impedance-based readout, but became much more obvious in case of RWG- or SPR-based assays. By contrast, the co-expression of hH1R and hMSR1 in HEK 293T cells strongly enhanced the signal compared to hH1R expression alone. As the sensitivity of the optical readouts is confined to a distance of 100-200nm from the surface, depending on the wavelength of the incident light, this observation is in accordance with tighter adhesion of the co-transfectants, inducing a shorter distance between the cell membrane and the substrate. Combining ECIS and SPR, allowing for simultaneous registration of both signals for a single cell population, provided a direct correlation of both readouts, when H1R or β2-AR stimulation was investigated for the same cell populations. Cell adhesion was found to have a critical impact on the results of label-free cell monitoring, in particular when techniques based on evanescent electric fields are applied.
authors.affiliation: Institute of Pharmacy, University of Regensburg, D-93053 Regensburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Lieb
authors.affiliation: Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93053 Regensburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Michaelis
authors.affiliation: Institute of Pharmacy, University of Regensburg, D-93053 Regensburg, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Plank
authors.affiliation: Institute of Pharmacy, University of Regensburg, D-93053 Regensburg, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Bernhardt
authors.affiliation: Institute of Pharmacy, University of Regensburg, D-93053 Regensburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Buschauer
authors.affiliation: Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93053 Regensburg, Germany. Electronic address: Joachim.Wegener@ur.de.
authors.forename: J
authors.initials: J
authors.lastname: Wegener
mesh_terms: D000818:Animals; D002417:Cattle; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D055664:Electrochemical Techniques / Q000295:instrumentation; D004867:Equipment Design; D057809:HEK293 Cells; D006801:Humans; D008027:Light; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D012031:Refractometry; D015398:Signal Transduction; D020349:Surface Plasmon Resonance / Q000295:instrumentation
chemical_list: D043562:Receptors, G-Protein-Coupled
keywords: Cell-based assays; Dynamic mass redistribution (DMR); ECIS; G-protein-coupled receptors; Holistic; Label-free; SPR