Abstract
A step-by-step procedure is described for functionalizing the surface of a glass coverslip so that a single cell contacts distinct patterns of extracellular matrix and cell–cell adhesion proteins. This dual-micropatterned substratum is accomplished through a two-step process. First, extracellular matrix (ECM) is microcontact-printed onto a silanized glass surface using electron beam lithography, etched resist-coated wafers, and Polydimethylsiloxane (PDMS) stamps of differing geometries. Then, non-ECM-coated surfaces are incubated sequentially with biotin, NeutrAvidin, and biotinylated Protein A to attach Fc-cadherin fusion proteins, Fc, or PEG. Cells are seeded at low density onto the functionalized surface for single-cell analysis of protein recruitment/turnover and cellular motility.
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Acknowledgments
We thank Dr. Nicolas Borghi for the design of dual-patterned substrata [5] and the Stanford Nanofabrication Facility (SNF) for their expertise. ML was supported by the Cancer Biology Program Training Grant (5T32 CA009151-34), and work in the Nelson laboratory was supported by the NIH (GM035527) and NSF (EFRI-MIKS).
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Lowndes, M., Nelson, W.J. (2013). Fabricating Surfaces with Distinct Geometries and Different Combinations of Cell Adhesion Proteins. In: Coutts, A. (eds) Adhesion Protein Protocols. Methods in Molecular Biology, vol 1046. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-538-5_13
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DOI: https://doi.org/10.1007/978-1-62703-538-5_13
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-62703-538-5
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