4.4. Cell Viability Assay

Cell proliferation upon treatment with buparlisib and/or trametinib was studied using a resazurin assay, as previously described [6]. All cell lines were seeded at a density of 5 × 10³ cells/well in 200 μL culture medium in 96 well plates (Nunc, Roskilde, Denmark) and treated with buparlisib or trametinib (0, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 µM), or a combination of both these treatments for a period of 72 h. After treatment, 20 µl of 0.01 mg/mL resazurin (Sigma-Aldrich Inc., St. Louis, MO, USA) diluted in phosphate buffered saline (PBS) was added to each well and incubated for 4 h at 37 °C. The absorbance was measured at dual mode 560/590 using a scanning multi-well spectrophotometer (Victor 3 1420 multi-label counter, Perkin Elmer, Waltham, MA, USA). Each treatment was performed in triplicate (n = 6 per experiment per drug concentration). IC₅₀ concentrations were drawn from the results, i.e., the drug concentration at which 50% of the cell growth was inhibited.