Matrigel 50 μl well

Cell migration and invasion assays were performed using 24-well plates and 8 μm transwell inserts (Corning Life Science, Acton, MA, USA). For migration assays, tumor cells were suspended in 200 μl serum-free RPMI-1640 medium (4 × 10⁴ cells) containing either 2% BSA or 0.1 mM PA and cultured in the upper chamber. Fetal bovine serum-conditioned medium (10%) (700 μl) was added to the lower 24-well plates. For invasion assays, the inserts were coated with Matrigel (50 μl/well) (BD Biosciences) and kept in a humidified incubator at 37 °C with 5% CO₂ for at least 2 h before adding the cells, tumor cells were suspended in 200 μl serum-free RPMI-1640 medium (1 × 10⁵ cells) containing either 2% BSA or 0.1 mM PA and cultured in the upper chamber. After a period of culture, tumor cells remaining in the upper side of the inserts were removed with cotton swabs. Tumor cells that migrated to the lower side of the inserts were fixed in methanol and stained with 0.5% crystal violet for 30 min at room temperature. Migrated cells were photographed using Nikon Digital Sight DS-U2 (Nikon, Tokyo, Japan) and Olympus BX50 microscopes (Olympus Optical Co. Ltd., Tokyo, Japan). Five visual fields were randomly chosen to calculate the number of migrated cells.

The tumor cells (3.0 × 10⁴) were seeded into 24-well format transwell chambers with 8.0um pore polycarbonate filter inserts (Costar, Cambridge, MA, USA). The chambers were placed in 24-well plates containing RPMI-1640 medium and 10% FBS. For migration assays, cells were suspended in 200 μl serum-free RPMI 1640 medium and cultured in the upper chamber. The lower chambers were filled with 700 μl RPMI-1640 and 10% FBS. For invasion assays, the inserts were coated membranes with Matrigel (50 μl/well) (BD Biosciences, Lake Franklin, NJ, USA) before adding the cells. Cells were incubated at 37°C for 24 h. Subsequently, filters were fixed using methanol for 15 min, stained with 0.1% crystal violet for 15 min, and washed twice with PBS. The number of cells were counted in five randomly selected microscopic views (100x).