4.8. Cell Invasion Assay

A trans-well membrane (Corning Costar, Cambridge, MA, USA) was coated with 30 μL Matrigel^® and incubated for 4 h and then CAL27 and Ca9-22 cells were seeded. Subsequently, the cells were treated with 0.5 mM polydatin and cultured for 48 h. The medium applied to the upper chamber of the Matrigel^®-coated trans-well was a serum-free medium containing 0.5 mM polydatin, and the medium applied to the lower chamber was filled with a medium containing 10% FBS, but not polydatin. The cells in the upper chamber were washed with PBS and fixed with 100% methanol for 10 min. The cells were stained with hematoxylin–eosin (H&E) for 30 min. After the membrane in the upper chamber was separated, it was washed three times in PBS, dehydrated through immersion in 70%, 80%, 90%, and 100% ethanol, mounted using malinol, and then photographed and analyzed using a Lionheart FX Automated Microscope. In the total area, the area of cells that penetrated the tran-swell and invaded on the membrane was digitized with Adobe Photoshop CS (San Jose, CA, USA) and expressed as a graph.