Transwell assay for the detection of cell invasion

Matrigel was dissolved overnight at 4°C and diluted with serum-free medium (SFM) (Thermo Fisher Scientific, US) at a ratio of 1:7. Matrigel invasion chambers were placed in the wells of a sterile 24-well plate, and 20 μL Matrigel was added into the chamber (ensuring that it evenly covered the chamber bottom). Following transfection (24 h), single-cell suspensions (1 × 10⁵ cells/mL) were produced in serum-free DMEM. The cells were added to the upper chamber of each well for transwell assay, with the 600 μL medium containing 10% fetal bovine serum (Thermo Fisher Scientific, US) in the lower chamber and maintained for 24 h at 37°C in a humidified incubator with 5% CO₂. The media was removed from the upper chamber, and cells that did not penetrate the membrane were removed using a wet cotton swab. The chamber was washed three times with PBS and fixed with 4% paraformaldehyde for 30 min. Crystal violet staining was performed (20-min incubation), and the chambers were imaged using an inverted microscope. Five visual fields were randomly selected, and the number of cells penetrating the stromal membrane were counted.