MTT assay for doubling time evaluation

MTT assay kit was purchased from Sigma (St. Louis, MO, USA) and used according to the standard protocol. The P4-cells were seeded at the density of 2×10⁴ cells/well in sterile 96-well plates containing 200 µl of DMEM+10% FBS. Cell division was evaluated in the presence of various concentrations of Dep (purchased from Zahravi Pharmaceutical Co., Tabriz, Iran) ranging from 10^–6 to 10^–10 M for 24 and 48 hours. Then, the supernatant was discarded and replaced with 100 µl fresh medium plus 10 µl MTT solution (5 mg/ml; Sigma), and cells were incubated at 37°C for 4 hours. Finally, 85 µl of the medium was replaced with 50 µl of DMSO and incubated for more than 10 minutes. Optical density was measured by an ELISA reader (Biotek, Winooski, VT, USA) at 540 nm. The experiment was repeated five times for each group.