Transwell Assay

Migration assay: PC cells in the logarithmic growth stage were starved for 24 h, collected and re-suspended to adjust the final cell concentration at 2 × 10⁵/ml. Then, 0.2ml suspension was added to the upper chamber of Transwell, and 700 μl pre-cooled DMEM cell culture solution containing 10% FBS was added to the lower chamber. Cells were cultured in a cell incubator containing 5% CO₂ at 37°C. After 24 h of incubation, the Transwell chamber was removed, cells on the upper chamber and basement membrane were wiped with a wet cotton swab, fixed with methanol for 30 min, and stained with 0.1% crystal violet for 20 min. After rinsing with running water, turning upside down, cells were dried naturally, and then observed and photographed under inverted microscope. The experiment was repeated three times.

Invasion assay: ECM glue (MERCK) was placed at 4°C overnight, which was then diluted the next day (pre-cool all gun heads and chambers on ice for half an hour before the experiment) with serum-free medium at the ratio of 1:9 to the final concentration of 1mg/ml. An amount of 40 μl ECM glue was added to the polycarbonate film of each 24-well Transwell upper chamber, and incubated in an incubator containing 5% CO₂ and at 37°C for 5 h, so that the ECM glue could polymerize to form a gel. After absorbing and discarding the superfluous liquid, pure DMEM medium was added 70 μl per chamber and incubated in 37°C incubator for 0.5 h, followed by rehydration of the matrix glue, and removal of the superfluous medium for further usage. Cells in each group were digested, centrifuged and re-suspended in DMEM medium without FBS for 24 h after serum withdrawal and starvation. The final cell concentration was adjusted to 2.5 × 105/ml. Afterward, 0.2 ml suspension was collected and added to the upper chamber where the basement membrane had been hydrated, followed by the addition of 700 μl pre-cooled DMEM medium containing 10% FBS to the lower chamber. It was cultured at 37°C in a 5% CO₂ saturated humidity incubator for 24 h. The chamber was removed, and the cells were removed from the chamber and basement membrane with a wet cotton swab, fixed with methanol for 30 min, stained with 0.1% crystal violet for 20 min, and dried upside down. Consequently, cells were observed and photographed under inverted microscope. The experiment was repeated three times.