Imaging and quantitative analysis of integrin-dependent cell-matrix adhesions

• Published in: STAR protocols 2023-09, Vol.4 (3), p.102473-102473, Article 102473
• Main Authors: van Stalborch, Anne-Marieke D., Clark, Andrew G., Sonnenberg, Arnoud, Margadant, Coert
• Format: Article
• Language: English
• Subjects: Cell Biology; Cell Culture; Cell Culture Techniques; Cell-Matrix Junctions; Embryonic Development; Female; Humans; Integrins; Microscopy; Pregnancy; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102473

Integrin-dependent cell-extracellular matrix adhesion is essential for wound healing, embryonic development, immunity, and tissue organization. Here, we present a protocol for the imaging and quantitative analysis of integrin-dependent cell-matrix adhesions. We describe steps for cell culture; virus preparation; lentiviral transduction; imaging with widefield, confocal, and total internal reflection fluorescence microscopy; and using a script for their quantitative analysis. We then detail procedures for analyzing adhesion dynamics by live-cell imaging and fluorescence recovery after photobleaching (FRAP). For complete details on the use and execution of this protocol, please refer to Margadant et al. (2012),1 van der Bijl et al. (2020),2 Amado-Azevedo et al. (2021).3 [Display omitted] •Confocal microscopy of cell-matrix adhesions and analysis of integrin traffic•Macro for automated quantitative analysis of cell-matrix adhesions•Live imaging of adhesion dynamics by widefield, confocal, and TIRF microscopy•Imaging and analysis of hemidesmosome behavior by FRAP Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Integrin-dependent cell-extracellular matrix adhesion is essential for wound healing, embryonic development, immunity, and tissue organization. Here, we present a protocol for the imaging and quantitative analysis of integrin-dependent cell-matrix adhesions. We describe steps for cell culture; virus preparation; lentiviral transduction; imaging with widefield, confocal, and total internal reflection fluorescence microscopy; and using a script for their quantitative analysis. We then detail procedures for analyzing adhesion dynamics by live-cell imaging and fluorescence recovery after photobleaching (FRAP).