2.6. In vitro invasion assay

In the transwell chamber, 100 μl of matrigel were added, followed by shaking and incubation at 37 °C for 4 h. After solidifying, 600 μl of serum-free 1640 medium were placed in the upper and lower chambers, and the membranes were hydrated for 30 min. Osteosarcoma cancer cells, which were transfected for 24 h, were utilized as cell suspensions in serum-free 1640 medium. Next, 100 μl of cell suspension (1 × 10⁴ cells) were placed in the upper chamber, and 600 μl of 10% FBS were pipetted into the bottom chamber. After 24 h, the residual cells (upper chamber) were removed and the chamber was washed with PBS. Subsequently, the cells were fixed with paraformaldehyde for 15 min, dyed with 0.1% crystal violet for 5 min, and washed with PBS. A microscope was used to observe the cells and count the cell numbers. In addition, transwell migration assay was performed using the same protocol as the invasion assay, except without a Matrigel pre-coating.