8 m transwell filters

The 8-µm Transwell filters (BD Biosciences) and 24-well Transwell chambers were used to detect the invasive capacity of cells. In total, 70 µl 1 mg/ml Matrigel (BD Biosciences) was added onto the upper chamber at 37°C overnight. Then, the upper chamber with Matrigel-coated membrane was seeded with 4×10⁴ NOZ cells in 200 µl serum-free medium. Moreover, 500 µl basal medium containing 15% FBS was added into the lower chamber. Following the 20-h co-culturing in an incubator containing 5% CO₂ at 37°C, the cells that invaded to the lower layer were fixed using 4% paraformaldehyde for 20 min and were then stained using crystal violet for another 20 min, both at room temperature. In total, five random fields were chosen to count the invaded cells using a light microscope (magnification, ×20) in order to determine the invasive capacity of NOZ cells. The assays were carried out in triplicate.

Cell motility was determined by cell invasion assays using 8-µm Transwell filters (BD Biosciences) with Matrigel (BD Biosciences). The treated ECA109 and TE-10 cells at a density of 5×10⁴ were seeded into the upper chamber with medium that did not contain serum; medium containing serum was placed into the lower chambers as a chemoattractant. After incubation for 48 h, the treated cells on the upper surface of the membrane were stained with crystal violet (0.1%) at 25°C for 10 min followed by manual counting under a microscope (magnification, ×20; Olympus Corp.) in three randomly selected areas. The Transwell assays were assessed using MShot Image Analysis System (Guangzhou Mingmei Photoelectric Technology Co., Ltd.). All experiments were repeated three times.