8 μm pore size polycarbonate membranes

The migration assay was conducted in 24-well Transwell chambers with 8‐μm pore size polycarbonate membranes (BD Biosciences, Franklin Lakes, NJ, U.S.A.), but invasion assay conducted in Transwell chambers with Matrigel (BD Biosciences, Franklin Lakes, NJ, U.S.A.) coated membranes. Briefly, transfected gastric cancer cells (5 × 10⁴ cells/well) were seeded with serum-free medium into the upper chambers, and medium with 15% FBS was added into the bottom chambers. After 24-h incubation, gastric cancer cells at the upper surface of membranes were removed, and gastric cancer cells penetrating to the lower side of membranes were fixed with methanol for 30 min and stained with 0.1% crystal violet solution for 20 min. Finally, the cell numbers were counted in five randomly chosen microscopic fields per membrane. These experiments were repeated three times.

Trans-well assay was done in 24-well Boyden chambers with 8 μm pore size polycarbonate membranes (BD Biosciences, San Diego, USA). ST1 cells (5 × 10⁴) were resuspended in 500 μl serum-free Medium 199 and placed in the upper chamber. The lower chamber was filled with serum-free Medium 199 with 750 μl CM or 400 nM final concentration of recombinant protein dissolved in an incomplete medium (serving as a chemoattractant). After 24 hr of incubation, cells remaining on the upper surface of the membrane were removed with a wet cotton bud. The cells that migrated to the lower surface of the membrane were fixed in ice-cold methanol, stained with crystal violet, and imaged in a light microscope.