4.6. Migration and Invasion Assays

A 24-well plate with 8-μm pore size chamber inserts (Corning, New York, NY, USA) was utilized, according to the manufacturer’s instructions, for cell migration assays and invasion assays. For migration assays, we seeded 5 × 10⁴ OS cells in the upper chamber in 200 μL serum-free DMEM (for MNNG/HOS cells) or RPMI-1640 (for U2OS cells) per well. For invasion assays, we seeded 1 × 10⁵ OS cell in the upper chamber in 200 μL serum-free DMEM (for MNNG/HOS cells) or RPMI-1640 (for U2OS cells) per well with the Matrigel-coated membrane. Eight hundred microliters of DMEM (for MNNG/HOS cells) or RPMI-1640 (for U2OS cells), supplemented with 10% fetal bovine serum, were added in the lower chamber. Non-migrated or non-invaded cells remaining at the top surface of the inserts were removed after incubation for 13 h or 16 h at 37 °C. The cells adherent to the lower surface of the inserts were fixed and stained with 0.1% crystal violet. Cells were counted and imaged through a CKX41 inverted microscope (Olympus, Tokyo, Japan). The assay was repeated three times.