Transwell Assay for Cell Migration

We seeded 5 × 10⁴ GI-ME-N cells per insert (8 µm, ThinCert™ Greiner Bio-One) in 200 µl 1% FBS-supplemented media containing DMSO or VPF. In the lower chamber, 800–1000 µl of 10% FBS media were used as a chemoattractant. The cells were allowed to migrate for 22 h, after which a cotton swab was used to remove the non-migrating cells from the upper chamber. The inserts were then washed in PBS before fixation for 20 min, at RT in 4% PFA solution. Following repeated washing steps for PFA removal, the migrating cells were stained for 30 min with 0.1% Crystal violet solution (Chroma-Gesellschaft Schmid & Co.). The inserts were washed and left to air-dry before being imaged using a ZEISS AxioImagerM2 fluorescence microscope. For the quantitative measurement of migrating cells, the crystal violet dye was extracted by incubating the membranes in a 10% acetic acid solution for 20 min, on a rocking platform at RT. The extracted dye was then spectrophotometrically measured at 595 nm. The transwell migration assay was performed in technical duplicates and repeated at least three times.