Biocoat control inserts with 8.0 m pet membrane

Transwell cell migration assay was performed using BioCoat Control Inserts with 8.0 µm PET Membrane (Corning, New York, NY, USA). LX2 (1 × 10⁴ cells) or HepG2 (2.5 × 10⁴ cells) were seeded onto the control insert in serum-free medium with or without recombinant FGF9 at a dose of 1 or 10 ng/ml. Cells were incubated with medium containing 2% FBS in the lower chamber for 24 hours. After removal of non-migrating cells on the upper surface of the membrane using cotton swabs, the cells migrated into the lower surface of the membrane were stained with Diff-Quik (Sysmex, Hyogo, Japan) and counted using a microscope in nine visual fields of each sample.

Transwell invasiveness assay was performed using Corning^® BioCoat™ Matrigel^® Invasion Chambers with 8.0 µm PET Membrane (Corning Incorporated, Corning, NY, USA). Transwell migration assay and control for invasiveness assay were done using Corning^® BioCoat™ Control Inserts with 8.0 µm PET Membrane (Corning Incorporated, Corning, NY, USA). Cells were previously starved in media supplemented with 2% FBS for 48 h and then seeded in concentrations HCT-116 (5 × 10⁴ cells/well), RKO (7.5 × 10⁴ cells/well), and HCT-15 (5 × 10⁴ cells/well) and incubated for 24 h in 5% CO₂ at 37 °C. Staining was performed with Hoechst 33,342 (Invitrogen), and migrated/invaded cells were visualized using a confocal microscope ZEISS LSM 700 and ZEISS ZEN Software (Carl Zeiss AG, Oberkochen, Germany). Subsequently, cells were counted using ImageJ software (Wayne Rasband, NIH, Bethesda, MD, USA).