Cell viability assay

To measure the effect of Metformin and Chrysin on cell growth, MTT assay was used with breast cancer (T47D) cell lines. For this assay, 6.5× 10³ cells/well were seeded in a 96-well plates and cultured. The cells were treated with different concentrations (0-120 µM) of free Metformin and free Chrysin after 24 h and equivalent doses of mixed Metformin-Chrysin for different time intervals (24–72 h). The cells grown in media containing equivalent amount of ethanol without Metformin and Chrysin served as control. After the treatment, media containing Metformin, Chrysin and mixed Metformin-Chrysin were removed carefully and 50 μL of 2 mg/mL MTT dissolved in phosphate buffered solution was added to each well, and the plate was coated with aluminum foil and incubated for 4 h. Then, contents of all wells were removed and 200 μl of pure DMSO were added to the wells followed by adding 25 μL Sorensen’s glycine buffer to each well. All experiments were set up in triplicates. Finally, the optical density (OD) was measured at 570 nm ELISA-reader with a reference wavelength of 630 nm. Results were expressed as mean ± S.D. IC50 (inhibitory concentration at 50%) was evaluated by Graph Pad 6 (Prism) software. Then, 5×10⁵ cells were treated with serial concentrations of pure Metformin and Chrysin and mixed Metformin-Chrysin (10, 20, 30, 40, and 50 µM). The same volume of 10% DMSO without Metformin and Chrysin was added to the flask containing control cells. The cells were then incubated at a temperature of 37 °C in a humidified incubator with a 5% CO₂ atmosphere for a 24 h.