Flow cytometry assays

The percentage of apoptotic cells 24 h after doxorubicin treatment was measured by flow cytometry (Becton Dickinson, NJ, USA). Cell labeling was performed with 0.5 mg/ml of FITC Annexin V-conjugated fluorochrome and 0.5 mg/ml o of DAPI and incubated for 15 min in darkness at room temperature using an FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen^™). For cell cycle analysis, cells were kept on ice prior to fixation. After detaching the cells with trypsin, 2 × 10⁶ cells were added per cytometer tube. Cells were fixed with 1 ml cold 80% ethanol and incubated for 2 h with ethanol at −20 °C. Finally, the cells were re-suspended with 1 ml DAPI/TX-100 solution (Sigma, St. Louis MO, USA), and incubated for 30 min at room temperature before flow-cytometry analysis. The samples were filtered prior to acquisition in order to eliminate any cell aggregates. The wavelengths of excitation and emission for DAPI were 405 nm and 450 nm respectively.