electric cell substrate impedance sensing ecis zθ instrument  (Applied BioPhysics)

Journal: International Journal of Molecular Sciences

Article Title: pH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis

doi: 10.3390/ijms23031419

Figure Lengend Snippet: The OGR1 inhibitor blocked OGR1-associated downstream events in Caco-2 cells and human CD14+ monocytes. ( A ) OGR1 (GPR68) antibody 16H23L16 for immunocytochemical detection of OGR1 was verified in previously validated tools. Caco-2 clone stably overexpressing OGR1 (clone U1); negative control, Caco-2 vector control (VC) cells. Cells were examined by confocal microscopy (Leica, Germany). ( B – F ) Functional assays confirmed pH-dependent OGR1-mediated signalling. ( B ) U1 and VC cells were subjected to an acidic pH shift and intracellular inositol phosphate (IP) formation was measured. Data are presented as mean ± SD and statistical analysis was performed using one-way ANOVA. *** p < 0.001. ( C ) Activated GTPase RhoA was measured in U1 and VC cells upon an acidic pH shift. ( D , E ) Efficacy of the OGR1 inhibitor (GPR68-I) and the enantiomer of the inhibitor were measured using electric cell-substrate impedance-sensing (ECIS) technology. OGR1 clone U1 and VC grown to confluent monolayers were subjected to an acidic pH shift with or without the OGR1 inhibitor (0.5, 1.0, 2.5, 5, 10, 25 µM). Changes in resistance of cell monolayers were monitored in real time. Representative graph of eight independent experiments shown for the inhibitor or the enantiomer. ( F , G ) Human CD14+ monocytes from healthy volunteers and active IBD patients were subjected to an acidic pH shift, with or without the OGR1 inhibitor (0.5, 1.0, 2.5, 5 µM). IP1 production was measured from healthy volunteers ( n = 5) and active IBD patients ( n = 2); active IBD patient ( n = 1). Samples in ( F , G ) were read on the Tecan Infinite or Biotek Synergy instrument, respectively. Data are presented as mean ± SD and statistical analysis was performed using one-way ANOVA. * p < 0.05. IBD: inflammatory bowel disease; OGR1: ovarian cancer G-protein-coupled receptor 1; vector control: VC.

Article Snippet: Resistance and impedance of Caco-2 monolayers were monitored using electric cell-substrate impedance-sensing (ECIS) instruments (Applied BioPhysics, Troy, NY, USA) as previously described [ ].

Techniques: Stable Transfection, Negative Control, Plasmid Preparation, Confocal Microscopy, Functional Assay, Electric Cell-substrate Impedance Sensing