Transwell Assay for Cell Invasiveness

Cell invasiveness was studied using the Trans-well Cell Culture (12 mm diameter, 8.0-fim pore size) purchased form Corning Incorporated (New York, NY, USA), as previously described [12 (link)]. Briefly, 9 × 10⁴ PGS, ANXA1 KO MIA PaCa-2 cells, transfected or not with miR-196a-5p mimic 24 h before, were seeded on the matrigel coating and maintained at 37 °C in 5% CO₂-95% air humidified atmosphere. After 24 h, the Trans-well Cell Culture chambers were washed twice with PBS and fixed with 4% p-formaldehyde for 10 min, and then with 100% methanol for 20 min. Later, the fixed cells were stained with crystal violet (0.5% w/v in a v/v solution of 20% methanol/distilled water; Merck Chemicals, Darmstadt, Germany) for 15 min. Next, the chambers were washed again in PBS and cleaned with a cotton bud to remove crystal violet exceedance. All of the experimental points were treated with mitomycin C (10 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) to ensure the block of mitosis. The number of cells that had migrated to the lower surface was counted in twelve random fields using EVOS light microscope (10×) (Life technologies Corporation, Carlsbad, CA, USA).