4.7. Transwell Migration Assay

An established transwell migration assay, utilizing 24-well hanging inserts, fitted with an 8 µm pore size membrane (Merck Millipore, Darmstadt, Germany), was used to investigate CRC cell migration [62]. Additionally, 1 × 10⁵ serum starved cells were seeded in triplicate in media containing 1% FBS, onto the apical surface of each hanging insert, and placed into wells containing 10% FBS. Following treatment with HES (10 µg), H. polygyrus antigen (10 µg), or 1× PBS, the plates were incubated for 24 h. Post-incubation, the lower surface of the insert was fixed with methanol and stained with crystal violet. Excess crystal violet was washed off the insert using distilled water before release of the crystal violet stain into 50% acetic acid. The absorbance of this solution was then quantified at 595 nm using a Rayto RT-2100C Microplate Reader (Rayto, Shenzhen, China).