PA and silicone gels were cast in glass-bottom dishes at a thickness of 500 μm and functionalized with fibronectin previously labeled with ATTO 488-NHS ester according to the manufacturer’s protocol (ATTO-TEC GmbH). Steel balls of 254, 175, and118 μm in radii (Abbott Ball Company) were coated with hydrophilic coatings (Coatings2Go) following the manufacturer’s protocol to minimize ball-substrate adhesion. Substrates coated with ATTO 488-labeled fibronectin were submerged in PBS and then indented by the steel balls. Surfactant-treated substrates were prepared by incubating the substrates with 1% Triton X-100 at room temperature for 1 hour, followed by five rinses with PBS before measurement. For indentation measurements of substrates with cultured cells, gels seeded with MCF10A cells at 5000 cells/cm2 and cultured for 24 hours before indentation measurements in media. For indentation measurements of substrates without adsorbed fibronectin, gels were submerged in PBS with free ATTO 488 dye to provide contrast for imaging. Gel surface profiles were visualized by taking z stack images of ATTO 488-conjugated fibronectin or ATTO 488 PBS at the indented gel-buffer interface using a Zeiss LSM 880 confocal inverted microscope (i880) with a 10×, 0.45-NA (numerical aperture) objective.

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