2019 journal article
Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, (152).
author keywords: Biochemistry; Issue 152; cell adhesion; cellular spreading; oxidative stress; fibronectin; epithelial cells; ATM inhibition; ruffles; stress fibers; cell circularity
MeSH headings : Animals; Cell Adhesion / drug effects; Cell Adhesion / physiology; Cell Count / methods; Cell Culture Techniques / methods; Cell Line; Cell Line, Transformed; Cells, Cultured; Epithelial Cells / drug effects; Epithelial Cells / metabolism; Extracellular Matrix / drug effects; Extracellular Matrix / metabolism; Fibroblasts / drug effects; Fibroblasts / metabolism; Fibronectins / metabolism; Humans; Microscopy, Fluorescence / methods; Morpholines / pharmacology; Oxidative Stress / drug effects; Oxidative Stress / physiology; Pyrones / pharmacology
TL;DR:
A step-wise protocol is described that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro to examine a broad range of biological questions.
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UN Sustainable Development Goal Categories
3. Good Health and Well-being
(Web of Science)
Source: Web Of Science
Added: November 18, 2019
The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.