F8806

The protocol of PAA gel preparation followed a previous report (Wang and Pelham, 1998 (link)) with some modifications. In brief, the surface of a glass-bottom culture dish was processed by NaOH (0.1 M, 5 min), (3-Aminopropyl) trimethoxysilane (100%, 3 min), and glutaraldehyde (0.5%, 20 min) to facilitate the covalent attachment of PAA. The PAA gel was prepared by mixing ddH₂O (258 µl), acrylamide (40%, 17 µl), bis-acrylamide (2%, 12 µl), ammonium persulfate (10%, 1.5 µl), and N,N,N’,N’-Tetramethylethylenediamine (100%, 0.45 µl), and 15 µl of the mixture was then mixed with 1.4 µl fluorescent microspheres (F8809, F8806; Invitrogen), dropped onto a previously prepared coverglass, and flattened with another cover glass. The dish was then put onto the surface of 24°C water under lamp light for polymerization. 20 min later, the coverglass over the gel was removed and the gel was exposed to UV for activating PAA after adding sulfo-SANPAH solution (1 mM; 22589; Thermo Fisher Scientific). The solution was removed 10 min later, and the gel was washed with Hepes (50 mM) on a shaker for 30 min, three times. The gel-coated dish was used immediately after preparation.

Polyacrylamide (PA) gels with different stiffnesses (0.7kPa, 4.6kPa, 9.3kPa, 19.5kPa) were synthesized following the previous protocols [26 ]. In short, prepolymer mixtures of 40% acrylamide solution (Fisher BP1402), 2% bis-acrylamide solution (Fisher BP1404), 1 M HEPES (Sigma H6147) solution, and deionized water were prepared for the desired Young’s moduli, with 1% (v/v) 200 nm crimson fluorospheres (Invitrogen F8806) added for cell traction experiments. Mixtures were then polymerized by adding 0.6% (v/v) 1% ammonium persulfate (Bio-Rad 161–0700) solution and 0.4% (v/v) TEMED (Fisher BP150), followed by being quicky pipetted onto salinized glass culture dishes (MatTek P35G-0-20-C) and covered with a circular cover slip (Fisher 12-545-80) to produce ~100 µm thick gels. After polymerization, PA gels were activated with 0.5 mg ml⁻¹ sulfo-SANPAH (Thermo 22589) and cultured with desired ECM molecules to conjugate these ECM molecules.
Mayo cells were plated on laminin with media containing 2% serum to promote adhesion. UCSD cells could not adhere to laminin, collagen, fibronectin, except for Matrigel with no serum.