2.10. Transwell Migration assay

PC3 cell migration assay was performed using 24 well Transwell chambers with 8 μm pore (Millipore, Billerica, MA, USA). Approximately 1 × 10⁵ cells/well were seeded in RPMI with 10% FBS with antibiotics in the presence or absence of catalase and cells were treated with different concentrations (10, 25 or 50 μM) of NCX4040 for 24 h. RPMI containing 10% FBS and human epidermal growth factor (hEGF) (5ng/ml) were added to the bottom chamber. The cotton swab was used to remove the non-migrated cells on the upper surface of the transwell membrane. The migrated cells present in the bottom surface of the Transwell insert and wells of the 24 well plates were fixed with 4% paraformaldehyde and stained with crystal violet (0.05%) according to manufacture instructions. The cells were photographed under a microscope. The percentage of migrated cells were stained and counted in 3 randomly chosen fields for each chamber.