Scm005

Geltrex™ LDEV-free reduced growth factor basement membrane matrix (12–18 mg/mL concentration; Thermo Fisher Scientific, Waltham, MA, USA) containing primarily laminin, collagen IV, entactin, and heparin sulfate proteoglycans was used. It is similar in composition to Matrigel™, but with different proportions of constituents. As-received matrix (hitherto referred as G-100) was diluted with serum-free ReNcell complete medium to result in 75%, 50%, and 25% Geltrex™ solutions (hitherto referred as G-75, G-50, G-25). Complete medium is defined as maintenance medium (SCM005; EMD Millipore, Burlington, MA, USA) added with 20 ng/mL each of bFGF and EGF (Thermo Fisher Scientific). The calculated volume of solutions was pipetted onto a 24-well plate and AFM specific petri dishes, maintained on ice. The cover glass coated with Sigmacote® (Sigma-Aldrich, St. Louis, MO, USA)^{28 (link)} was used to uniformly spread the small volumes in the well-plate or petri dish to obtain the desired gel height. Briefly, coverslips treated with Sigmacote® were placed on the solutions which were allowed to gel (30 min, 37 °C), and the coverslips were removed to obtain a ~ 70 μm thick gel. Tissue culture polystyrene (TCPS; Thermo Fisher Scientific) dishes were functionalized with laminin (Sigma-Aldrich) for two hours prior to use.