3D Cell Invasion Assay with Growth Factors

3D cell culture assays were performed as previously described19 (link). 2 × 10⁴ cells per well were seeded on top of the matrix and left for invasion under standard conditions (37 °C, 5% CO₂) in DMEM/HAM’s F12 medium containing 5% FBS. Treatment of cells with recombinant epidermal growth factor (EGF) (Sigma), transforming growth factor beta-1 (TGFβ1) (R&D, Minneapolis, MN, USA)), fibroblast growth factor (FGF) 2 (R&D) or platelet derived growth factor (PDGF) BB (life technologies; Carlsbad, USA) was accomplished by treating cells with 10–50 ng/ml EGF and FGF2, 1–5 ng/ml TGFβ1, or 5–25 ng/ml PDGF-BB 24 hours after plating, and culturing them up to 72 hours in total.