Evos light microscope 10

A 24‐well plate was coated with Matrigel (Becton Dickinson Labware, Franklin Lakes, NJ, USA) mixed to EGM‐2 1 : 1 for HUVEC cells and MLEC medium 1 : 1 for MLEC WT and Sdc4‐/‐ on ice and incubated at 37 °C for 30 min to allow gelation to occur. The cells were seeded to the top of the gel at a density of 2 × 10⁴ cells/well in presence or not of the treatments. Cells were incubated at 37 °C with 5% CO₂. After 12 h, pictures were captured using EVOS^® light microscope (10×) (Life technologies Corporation). The length of each tube was measured, and the number of branches was calculated using imagej (NIH, Bethesda, MD, USA) (Angiogenesis Analyzer for imagej) software.

A 24-well plate was coated with Matrigel (Becton Dickinson Labware, Franklin Lakes, NJ, USA) mixed to EGM-2 1:1 on ice and incubated at 37 °C for 30 min to allow gelation to occur. As reported in [47 (link)], HUVEC cells were added to the top of the gel at a density of 2 × 10⁴ cells/well in the presence or not of the indicated treatments. Cells were incubated at 37 °C with 5% CO₂. After 12 hours, pictures were captured using EVOS^® light microscope (10×) (Life technologies Corporation, Carlsbad, CA, USA). The length of each tube was measured and the number of branches was calculated using ImageJ (NIH, Bethesda, MD, USA) (Angiogenesis Analyzer for ImageJ) software.

A 24-well plate was coated with Matrigel (Becton Dickinson Labware, Franklin Lakes, NJ, USA) mixed to EGM-2 1:1 on ice and incubated at 37 °C for 30 min to allow gelation to occur. HUVEC cells were added to the top of the gel at a density of 2 × 10⁴ cells/well in the presence or not of sodium mesoglycan and Prisma^® Skin. Cells were incubated at 37 °C with 5% CO₂. After 12 h, pictures were captured using EVOS light microscope (10×) (Life technologies Corporation, Carlsbad, CA, USA). The length of each tube was measured and the number of branches was calculated using ImageJ (NIH, Bethesda, MD, USA) (Angiogenesis Analyzer for ImageJ) software.

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).

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).

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).