Cancer-associated fibroblasts (CAFs) and the osteosarcoma cell line Saos-2 (ATCC® HTB-85) were used to produce cell-derived extracellular matrices. Matrices were prepared, as described previously (Oliver-Kozup et al., 2013 (link)). Briefly, CAFs and Saos-2 cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C in an atmosphere of 5% CO2 throughout the experiment. To produce the extracellular matrix for specific tests, cells were grown as follows: (i) for matrix characterization via immunofluorescence or for GFP expressing GAS (GFP-GAS) attachment assays, cells were grown on 15-mm glass coverslips inserted into wells of (24-well) tissue culture plates, and (ii) for crystal violet biofilm assay, matrix characterization by ELISA or by Ponceau S staining, cells were growth in plastic wells without glass coverslips. Cells were seeded at 50,000 cells per well, grown until confluent, and then detached through treatment with 5 mM ethylene glycol tetraacetic acid (EGTA) and removed from wells. Samples were washed gently with PBS and wells or coverslips were subsequently used for assessment.