4.1. Cell Culture

The human colon carcinoma cell line HT29 was obtained from the American Type Culture Collection and was grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, glutamine (4 mM), and penicillin (100 U/mL)/streptomycin (100 g/mL). Cell cultures were maintained at 37 °C in 5% CO₂ and 95% air. HT29 cells were plated at the approximate density of 1 × 10⁶ cells/dish in 10 cm² culture dishes, 4 × 10⁴ cells/well in two-well chamber glass slides, and 1 × 10⁴ cell/well in 96-well tissue culture plates. When cells reached approximately 60% confluence, the medium was replaced with serum-free cell medium containing different concentrations of metformin (0, 10, 25, and 50 mmol/L) and the cells were cultured for 24 and 48 h at 37 °C in 5% CO₂ and 95% air. The cells were trypsinized with 0.25% trypsin and 0.2 g/L EDTA, and harvested for further studies. Metformin was purchased from Sigma Aldrich (Saint Louis, MO, USA; PHR 1084) and was dissolved in sterile water at a stock dose of 1 M for further application.