Analyzing Cell Invasiveness through Trans-well Assay

Cell invasiveness was analyzed as reported in Belvedere et al., 2014 [27 (link)]. Briefly, the upper front of trans-well Cell Culture (12 mm diameter, 8.0-fim pore size; Corning Incorporated) was coated with Matrigel (Becton Dickinson Labware), diluted with 3 volumes of serum-free medium and stored at 37 °C until its gelation. Cells were plated in 350 µL of medium serum-free at a number of 4 × 10⁴/insert in the upper chamber of the trans-well. Then, 1.4 mL of supplemented growth medium with or without sodium MSG or PCL-derived MSG at 24, 48, and 72 h were put in the lower chamber and the trans-well was left for 24 h at 37 °C in 5% CO₂-95% air humidified atmosphere. After that, the medium was aspirated, the filters were washed twice with PBS 1× and fixed with 4% p-formaldehyde for 10 min, then with 100% methanol for 20 min. The filters so fixed, were stained with 0.5% crystal violet prepared from stock crystal violet (powder, Merck Chemicals, Darmstadt, Germany) by distilled water and 20% methanol for 15 min. After that, the filters were washed again in PBS 1× and cleaned with a cotton bud. The number of migrated cells to the lower surface was counted in twelve random fields using an EVOS light microscope (10×) (Life technologies Corporation, Carlsbad, CA, USA).