3.4.3. Cell Treatments

GES-1 cells were seeded in a 24-well plate (Falcon^®, Corning Life Science, Amsterdam, The Netherlands) at a density of 3 × 10⁴ cells/well or in a 12-well plate at a density of 6 × 10⁴ cells/well, according to the specific investigation. After 72 h, GES-1 cells were treated with the pro-inflammatory stimulus TNFα (10 ng/mL) or H. pylori (bacterium-to-cell ratio of 50:1) for 1 or 6 h, along with the extracts at different concentrations. In the co-culture, serum starvation was performed the day before the treatment to allow the synchronization of cells, using 0.5% serum medium, supplemented with 1% L-glutamine and 1% penicillin/streptomycin. The treatments in the co-culture approach were conducted with serum- and antibiotic-free medium, whereas TNFα treatments were carried out in serum-free medium. During the treatment, cells were maintained in an incubator at 37 °C and 5% CO₂.