Mtt based in vitro toxicology assay

AML cells were treated with compound diluent only (controls) or with the MCL1-inhibitor S63845 (HY-100741, MedChemExpress, Monmouth Junction, NJ, USA), the MDM2-inhibitor NVP-HDM201 (Novartis, Switzerland), and the MEK1/2-inhibitor trametinib (HY-10999A, MedChem Express, Monmouth Junction, NJ, USA). Cell viability was determined using the MTT-based in vitro toxicology assay (SIGMA-ALDRICH, St. Louis, MO, USA). For AML cell lines, four independent assays (biological replicates) with four measurements (technical replicates) per dosage were performed. For hematological patient samples two independent assays with three technical replicates per dosage were performed. Data were analyzed on GraphPad Prism software using Mann–Whitney tests and are depicted as average values with standard deviation on column graphs.

Cell viability was determined using a commercial MTT-based in vitro toxicology assay (Sigma-Aldrich), which detects viable cells colorimetrically based on the production of purple formazan. MRC-5 and MLE12 cells were initially seeded in 96-well plates at a density of 2 × 10⁴ cells/well and 3 × 10⁴ cells/well, respectively, prior to incubation for 24 h at 37 °C. Cell culture media were replaced by complete media containing the indicated concentration of HSM ethanol extract, prior to incubation for 24 h or 48 h. After incubation, 10 μl of MTT (5 mg/ml) were added to each well, and the plates were incubated for 4 h at 37 °C. The content of each well was eluted and the precipitate was dissolved in 100 μl of the MTT solubilization solution. Absorbance was read at 570 nm using a SpectraMax M5 multi-mode microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability (%) was calculated as the ratio of surviving cells in the HSM-treated group divided by that of the control group. All treated samples and controls were tested in triplicate.