Transwell Migration Assay Protocol

A transwell migration system utilizing 8.0 µm pore polyethylene terephthalate (PET, BD Falcon, MA, USA) inserts was used for the migration experiments as we previously described^{29 (link)}. Briefly, inserts were placed in 12-well notch plate, and 5 × 10⁴ cells were suspended in DMEM supplemented with 1.0% serum and were seeded in the upper chamber, while DMEM supplemented with 10% serum was used as attractant in the lower chamber. Twenty four hours later, non-migrating cells were gently removed using cotton swab, while migrated cells adherent to the lower surface of the membrane were stained with Diff-Quik Staining Kit (Siemens Healthcare Diagnostics, USA). The number of migrated cells was counted in six different random fields using bright-field microscope (Axio Observer-A1, Carl Zeiss, Germany) at 5X magnification.