2.9. Cell migration and invasion analysis

In brief, 200 μL Keratinocyte-Serum Free Medium containing the transfected cholesteatoma keratinocytes (1 × 10⁵ cells) was added to the upper chamber of the transwell (Costar, Cambridge, MA). The bottom of the transwell chamber was supplemented with 600 μL Keratinocyte-Serum Free Medium containing FBS (10%, Sigma). The migrated and invaded cells were fixed with paraformaldehyde (4%, Sigma) and stained with crystal violet (0.25%, Solarbio). The upper chamber of the transwell used in the invasion assay was pre-coated with matrigel (Sigma), but the migration assay did not. An inverted microscope (MTX Lab Systems) at 100× magnification was applied to calculate and photograph the migrated and invaded cells.