Upper chamber

Approximately 5 × 10³ cells were suspended in 200 μl of serum-free media and cultured in the upper chamber (8 μm pore size, Millipore) for the migration assay. For the invasion assay, the chamber was coated with 20% Matrigel (Sigma) 24 hours before culturing cells. Approximately 1 × 10⁴ cells were suspended in 200 μl of serum-free media and cultured in the upper chamber (8 μm pore size, Millipore), and 800 ml of the cell growth medium was added to the lower chambers. After a 48-hour incubation, the cells that had migrated or invaded through the membrane from the upper chamber to the lower chamber were fixed by 4% paraformaldehyde and stained with 0.1% crystal violet (Sigma). The cells below the chamber surface were photographed, and the cells counted in three random fields.

Cell migration and invasion were determined using 24-well transwell plates (Corning Inc., USA) with 8-μm-pore polycarbonate membranes. Matrigel (BD Biosciences, USA) was thawed at 4°C overnight and diluted with serum-free RPMI-1640 medium (dilution 1:2). The mixture (50 μL) was evenly smeared into the upper chamber (Merck Millipore, USA) and the chambers were incubated at 37°C for 1 h. After solidification, 1×10⁵ cells from each group were seeded into the upper chamber containing 200 μL serum-free RPMI-1640 medium. Next, 500 μL RPMI-1640 medium supplemented with 10% fetal bovine serum was added into the lower chamber. After 24 h, the chamber was removed and the cells in the upper chamber were wiped off. After fixation with 4% formaldehyde for 10 min, the membrane was stained using the Giemsa method for microscopic observation. The number of migrated cells was calculated and averaged in five random fields (200×). All procedures were carried out on ice with pipetting tips precooled at 4°C.

Growth factor-depleted Matrigel invasion chambers (BD Biosciences, Franklin Lakes, NJ, USA) were used to measure cell invasion. Matrigel was thawed at 4 °C overnight and diluted with serum-free DMEM (dilution 1:2). The mixture (50 μl) was evenly distributed into the upper chamber (Merck Millipore, Billerica, MA, USA) and incubated at 37 °C for 1 h. After solidification, 1 × 10⁵ cells from each group were seeded into the upper chamber containing 200 μl of serum-free DMEM. In addition, 500 μl DMEM supplemented with 10% fetal bovine serum was added to the lower chamber. After 24 h, the chamber was removed, and the cells in the upper chamber were wiped off. After being fixed with 4% formaldehyde for 10 min, the membrane was stained using Giemsa method for microscopic observation of five random fields (200×). For migration array, the tumor cells were seeded onto the upper chamber (Merck Millipore, Billerica, MA, USA) without matrigel, and the rest of the steps were the same as the invasive array. The number of motile cells was calculated to evaluate cell invasion and migration. All procedures were carried out on ice with pipetting tips being cooled to 4 °C.