RRC ID 61970
Author Keselowsky BG, García AJ.
Title Quantitative methods for analysis of integrin binding and focal adhesion formation on biomaterial surfaces.
Journal Biomaterials
Abstract Integrin binding and focal adhesion assembly are critical to cellular responses to biomaterial surfaces in biomedical and biotechnological applications. While immunostaining techniques to study focal adhesion assembly are well established, a crucial need remains for quantitative methods for analyzing adhesive structures. We present simple yet robust approaches to quantify integrin binding and focal adhesion assembly on biomaterial surfaces. Integrin binding to fibronectin and a RGD-containing synthetic peptide was quantified by sequentially cross-linking integrin-ligand complexes via a water-soluble homo-bifunctional cross-linker, extracting bulk cellular components in detergent, and detecting bound integrins by ELISA. Focal adhesion components (vinculin, talin, alpha-actinin) localized to adhesion plaques were isolated from bulk cytoskeletal and cytoplasmic components by mechanical rupture at a plane close to the basal cell surface and quantified by Western blotting. These approaches represent simple and efficient methodologies to analyze structure-function relationships in cell-material interactions.
Volume 26(4)
Pages 413-8
Published 2005-2-1
DOI 10.1016/j.biomaterials.2004.02.050
PII S0142961204001991
PMID 15275815
MeSH 3T3 Cells Animals Biocompatible Materials / chemistry Blotting, Western / methods* Cell Adhesion / physiology Cell Culture Techniques / methods* Cell Fractionation / methods* Cross-Linking Reagents / analysis Cross-Linking Reagents / metabolism Enzyme-Linked Immunosorbent Assay / methods* Focal Adhesions / physiology* Integrins / analysis Integrins / metabolism* Materials Testing / methods* Mice Protein Binding Reproducibility of Results Sensitivity and Specificity
IF 10.317
Human and Animal Cells MC3T3-E1(RCB1126)