title: Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.
abstract: The past 20 years has seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists and pharmacological and toxicological compounds. Most studies on barrier function use G protein-coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance-based techniques such as electric cell-substrate impedance sensing (ECIS) resides in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications, and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research, little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency, or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling, and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine, and sphingosine-1-phosphate.
authors.affiliation: The SUNY College of Nanoscale Science and Engineering (CNSE), SUNY Polytechnic Institute, State University of New York, 257 Fuller Rd., Albany, NY, 12203, USA.|Applied BioPhysics Inc., Troy, NY, USA.
authors.forename: Judith A
authors.initials: JA
authors.lastname: Stolwijk
authors.affiliation: Department of Physiological Sciences, East Virginia Medical School, Norfolk, VA, USA.
authors.forename: Khalid
authors.initials: K
authors.lastname: Matrougui
authors.affiliation: Applied BioPhysics Inc., Troy, NY, USA.
authors.forename: Christian W
authors.initials: CW
authors.lastname: Renken
authors.affiliation: The SUNY College of Nanoscale Science and Engineering (CNSE), SUNY Polytechnic Institute, State University of New York, 257 Fuller Rd., Albany, NY, 12203, USA. mtrebak@albany.edu.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Trebak
mesh_terms: D000465:Algorithms; D002478:Cells, Cultured; D017097:Electric Impedance; D004594:Electrophysiology / Q000379:methods*; D042783:Endothelial Cells / Q000378:metabolism / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D006801:Humans; D008564:Membrane Potentials; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism*
chemical_list: D043562:Receptors, G-Protein-Coupled
keywords: Barrier function; ECIS; Endothelial cells; Impedance; Resistance; TEER