Quantitative methods for analysis of integrin binding and focal adhesion formation on biomaterial surfaces.
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.
Keselowsky
Benjamin G
BG
Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
García
Andrés J
AJ
D016475:3T3 Cells; D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry; D015153:Blotting, Western / Q000379:methods*; D002448:Cell Adhesion / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002458:Cell Fractionation / Q000379:methods*; D003432:Cross-Linking Reagents / Q000032:analysis / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D022001:Focal Adhesions / Q000502:physiology*; D016023:Integrins / Q000032:analysis / Q000378:metabolism*; D008422:Materials Testing / Q000379:methods*; D051379:Mice; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001672:Biocompatible Materials; D003432:Cross-Linking Reagents; D016023:Integrins