Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Summary

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Abstract

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Introduction

Stable adhesive contacts between cells and their surrounding extracellular matrix are required for maintenance of tissue homeostasis. For example, endothelial cell adhesion to the subendothelial matrix in blood vessels plays a critical role in maintaining the integrity of the endothelial layer and its homeostatic function as a regulatory, semi-permeable vascular barrier¹. The actin cytoskeleton is mechanically coupled to adhesive matrix molecules at sites of cell-matrix adhesion and adhesive contacts at the cell-matrix interface play an important role in determining the position of the cell membrane by resisting centrally-directed actomyosin tensile forces.....

Protocol

1. General Endothelial Cell Culture

1. Culture bovine aortic endothelial cells (passages 4–9) on gelatin-coated tissue culture flasks (coat tissue culture surface with 0.1% w/v gelatin in PBS at RT for 15 min) in EGM-2 media (with the EGM-2 bullet kit containing 5% fetal bovine serum, growth factors and all supplements provided by the manufacturer, except for hydrocortisone).
2. When cells are near-confluent (ca. 3 days post seeding after a 1:4 split), harvest cells by treatment with 0.05% w/v trypsin / 0.02% w/v EDTA in PBS at 37 °C. After the majority of cells have detached, add complete EGM-2 media to quench the trypsin and then centrifug....

Results

Real-time quantification of endothelial cell de-adhesion from fibronectin in response to MPO-mediated fibronectin oxidation (Experiment 2). The seeding of endothelial cell suspensions onto native (MPO free) fibronectin or MPO-bearing fibronectin results in maximal cell attachment and spreading within 2 hr, as judged by a plateauing of cell index values in the cell substrate impedance measurements (Figure 3A). This initial phase of cell attachment and spreading is markedly reduced when MP.......

Discussion

Cell-matrix adhesion and de-adhesion processes can be quantified accurately in real time with high temporal resolution using cell-substrate impedance and live cell imaging analyses. These real-time approaches provide a major advantage over end-point analyses of cell adhesion, which provide poor temporal resolution. By accurately quantifying rapid de-adhesion responses with high temporal resolution, these analyses can provide critical insights into how morphological responses to matrix modifications are regulated and how .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 well gold cell-substrate impedance microelectrode array	ACEA Biosciences / Roche	E-Plate 96	single-use plate used for performing cell-based assays on the xCELLigence system
blebbistatin	Sigma	B0560	selective inhibitor of non-muscle myosin-II
Bovine Aortic Endothelial Cells, cryopreserved	Lonza	BW-6001
Bovine serum albumin	Sigma	05470
EGM-2 BulletKit	Lonza	CC-3162	endothelial cell growth media kit
Fibronectin, lyophilized powder	Sigma	F-4759	from bovine plasma
Fluorodish 35 mm glass-bottomed cell culture dish	World Precision Instruments	FD35-100	Cell cuture dish with optical quality glass bottom for imaging
Gelatin from bovine skin	Sigma	G9391	cell culture substratum
Hank's Balanced Salt Solution	Life Technologies	14025076
Hydrogen peroxide	Merck	107298
Medium-199	Life Technologies	11150-059	serum-free cell media
Methionine	Sigma	M9500	quenches chlorinating oxidants generated by myeloperoxidase
Myeloperoxidase, Human Polymorphonuclear Leukocytes	Millipore	475911
RTCA MP Instrument / xCELLigence cell substrate impedance system	ACEA Biosciences / Roche		Consists of RTCA Analyzer, RTCA (Multiple Plate) MP Station and RTCA Control Unit
Trypsin-EDTA (0.5%)	Gibco	15400-054