2.11. Transwell migration and invasion assays

Transwell migration assays were performed in a 24-well Transwell Inserts (Corning). HeLa cells (5 × 10⁴) suspended in 200 μl serum-free medium were seeded in the upper chamber, and 600 μl DMEM containing 1% FBS was placed in the lower chamber. After incubation for 24 h at 37 °C, the cells in the upper chamber were carefully removed with a cotton swab, and the cells that had migrated to the reverse face of the membrane were fixed in methanol and stained with 0.1% crystal violet. Five fields were randomly selected from the central and surrounding parts of the membrane under a microscope, and cells were counted and normalized as indicated for statistical analysis. Transwell invasion assays were performed similarly as the migration assay, except the membrane of the upper chamber was Matrigel (BD Biosciences) precoated to mimic extracellular matrix.