Trans well cell culture

MIA PaCa-2 and PANC-1 invasiveness was studied using the Trans-well Cell Culture (12 mm diameter, 8.0-fim pore size) purchased form Corning Incorporated (USA). The chambers were coated with Matrigel (Becton Dickinson Labware) that was diluted with 3 volumes of DMEM serum-free and stored at 37°C until its gelation. Cells were plated in 350 μl of DMEM serum-free at a number of 9 × 10⁴/insert in the upper chamber of the trans-well. 1,4 ml of DMEM with or without FBS were put in the lower chamber and the trans-well was left for 24 hours 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 minutes, then with 100% methanol for 20 minutes. The filters so fixed, were stained with 0,5% crystal violet prepared from stock crystal violet (powder, Merck Chemicals) by distilled water and 20% methanol for 15 minutes. After that, the filters were washed again in PBS 1× and cleaned with a cotton bud. 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).

AGS and AGS-AC1 invasiveness was analyzed using Trans-well Cell Culture (12 mm diameter, 8.0-μm pore size; Corning Incorporated; Corning, NY, USA). The membranes of the upper chambers were coated with Matrigel (Becton Dickenson, Franklin Lakes, NJ, USA) and placed in a well containing media supplemented with 10% FBS. The cells were seeded at a number of 9 × 10⁴/insert into the upper chambers in serum-free medium. Treatments with recombinant TFF1 protein (hrTFF1, Raybiotech, Norcross, GA, USA) and conditioned media were carried out in the lower chamber. After 24 h of incubation at 37 °C in 5% CO₂–95% air humidified atmosphere, the filters were fixed with 4% p-formaldehyde for 10 min and then with 100% methanol for 20 min. The cells on the lower surface of the filter were stained with a 0.5% crystal violet solution. The cells migrating to the lower surface were counted in twelve random fields using EVOS light microscope (10×) (Life technologies Corporation, Carlsbad, CA, USA).

MIA PaCa-2 invasiveness was studied using the Trans-well Cell Culture (12 mm diameter, 8.0-fim pore size) purchased form Corning Incorporated (USA), as previously described19 (link). Briefly, at 24 hours after seeding or treatments, the Trans-well Cell Culture chambers were washed twice with PBS and fixed with 4% p-formaldehyde for 10 minutes, then with 100% methanol for 20 minutes. Following, the fixed cells were stained with crystal violet (0, 5% w/v in a v/v solution of 20% methanol/distilled water; Merck Chemicals) for 15 minutes. After that, the chambers were washed again in PBS and cleaned with a cotton bud to remove crystal violet exceedance. The number of cells that had migrated to the lower surface was counted in twelve random fields using EVOS light microscope (10X) (Life technologies Corporation).

Cell invasiveness was studied using the Trans-well Cell Culture (12 mm diameter, 8.0-fim pore size) purchased from Corning Incorporated (New York, NY, USA), as previously described [20 (link)]. Briefly, 7 × 10⁴ HaCaT cells were plated in 350 µL of medium serum-free in the upper chamber of the trans-well. 1,4 mL of DMEM with FBS and with or without Ac2-26 and Boc-1 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 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).