Transwell permeable supports 6.5 mm insert

A total of 50,000 cancer cells were seeded into 24-well plates approximately 24 h prior to the start of the assay. HUVECs were serum-starved in DMEM supplemented with 0.2% FBS for 20 h. Cancer cells were washed with PBS and 1 ml of 0.2% FBS DMEM was added to each well. Each well was then fitted with a Transwell Permeable Supports 6.5 mm Insert (Costar). In all, 80,000 HUVECs, resuspended in 0.2 ml of starvation media, were seeded into each insert and incubated at 37 °C for 18 h.

The ability of PC cells to migrate or invade was verified in Transwell Permeable Supports (6.5 mm Insert; Costar, Cambridge, Mass). After 24 h of reaction, cells were resuspended in DMEM without FBS at 5 × 10⁴ cells/mL and seeded in the upper chamber at 5 × 10³ cells per well, while the lower well was filled with 500 μl of DMEM supplemented with 20% FBS. Then, the plate was placed at 37  °C with humidified 5% CO₂. After approximately 1 day of culture, the cells that migrated through the filter membrane were fixed with 4% polyformaldehyde for 20 min at room temperature, following by rinsing with PBS. Cells were stained for 20 min with 0.1% crystal violet at room temperature. Cotton buds were utilized to wipe away residual cells left on the lower cover of the chamber. Images were obtained with Image-Pro Plus (IPP) 6.0 after the above steps. For each treatment group, 3 individual fields of cells were counted to evaluate cell transferability. Cell invasion assays were performed following the same steps used to conduct the cell migration assay, with the only difference being that the bottom of the chamber was precoated with 10 μg/mL fibronectin gel.