Pflügers Archiv - European Journal of Physiology

, Volume 467, Issue 10, pp 2193–2218

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements

  • Judith A. Stolwijk
  • Khalid Matrougui
  • Christian W. Renken
  • Mohamed Trebak
Signaling and cell physiology

DOI: 10.1007/s00424-014-1674-0

Cite this article as:
Stolwijk, J.A., Matrougui, K., Renken, C.W. et al. Pflugers Arch - Eur J Physiol (2015) 467: 2193. doi:10.1007/s00424-014-1674-0

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.

Keywords

ECIS Endothelial cells Barrier function Impedance Resistance TEER 

Abbreviations

AC

Alternating current

BBB

Blood brain barrier

DC

Direct current

ECIS

Electric cell-substrate impedance sensing

FITC

Fluorescein-isothiocyanate

GPCR

G protein-coupled receptor

HDMEC

Human dermal microvascular endothelial cells

His

Histamine

HUVEC

Human umbilical vein endothelial cells

ITO

Indium tin oxide

RVSMC

Rat vascular smooth muscle cells

S1P

Sphingosine-1-phosphate

TEER

Transendothelial (/epithelial) electrical resistance

Thr

Thrombin

8W1E

8 Wells

1 Electrode per well

(1 Working electrode/1 counter electrode)

8W10E

8 Wells

10 Electrodes per well

(Working electrode consisting of 10 small electrodes/1 counter electrode)

8W10E+

8 Wells

40 Electrodes per well

(40 Electrodes distributed on interdigitated finger pattern)

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Judith A. Stolwijk
    • 1
    • 2
  • Khalid Matrougui
    • 3
  • Christian W. Renken
    • 2
  • Mohamed Trebak
    • 1
  1. 1.The SUNY College of Nanoscale Science and Engineering (CNSE), SUNY Polytechnic InstituteState University of New YorkAlbanyUSA
  2. 2.Applied BioPhysics Inc.TroyUSA
  3. 3.Department of Physiological SciencesEast Virginia Medical SchoolNorfolkUSA

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