Modified two chamber migration assay

Cell migration was determined using a modified two-chamber migration assay (Corning Incorporated, Corning, NY, USA) with a pore size of 8 µm. Cells (Ad-podoplanin CAFs and Ad-vector CAFs) were seeded in 1% FBS/Dulbecco's modified Eagle's medium (DMEM) in the upper chamber at a concentration of 2×10⁵ cells/ml, and the lower chamber was filled with 10% FBS/DMEM. After 24 h of incubation at 37°C, cells within the upper chamber were removed with a cotton swab. Cells which had migrated across the membrane were fixed in 4% paraformaldehyde at 37°C for 30 min, stained with crystal violet (0.5% in 20% methanol) at room temperature for 30 min. Stained cells were counted in five randomly selected fields using a light microscope (magnification, ×100; Olympus Corporation).

Cell migration was detected using a modified two-chamber migration assay (Corning Incorporated) with a pore size of 8 μm. Briefly, transfected or nontransfected OS-732 cells (1 × 10³) were resuspended in 200 μl of serum-free DMEM and added into the upper chamber. Complete DMEM (600 μl) was added into the lower chamber. After incubation at 37°C for 48 h, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) immediately. Nontraversed cells in the upper chamber were removed using a cotton swab, and traversed cells in the lower chamber were counted under a microscope (Nikon, Tokyo, Japan). Cell migration (%) was calculated by traversed cells in transfected group/traversed cells in nontransfected (control) group × 100%.
Cell invasion was measured similarly to that for cell migration except that the Transwell membrane was precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA).