Sytox green nucleic acid stain

Cells were plated and transfected in 6-well plates at 0.094 fmol siRNA per cell. At 24 hours post-transfection, cells were treated with the indicated doses of cisplatin. At the indicated time points, the supernatant and adherent cells harvested by trypsinization were collected. For cell cycle analysis, the cells were washed with HBSS and fixed in 70% ethanol at 10⁶ cells/mL for 1 hour on ice followed by an overnight incubation at −20°C. Cell pellets were collected by centrifugation and then stained in PBS buffer containing 50 μg/mL propidium iodide (Invitrogen), 1 mg/mL RNase A (Sigma), and 0.1% Triton X-100 (Bio-Rad) for 15 min at 37°C followed by 1 hour on ice. To assess DNA damage, transfected cells were treated with cisplatin or topotecan for 48 hours, and then fixed and stained with anti-γH2AX antibody and counter-stained with Sytox^®green nucleic acid stain according to manufacturer's instructions included in the Apoptosis, DNA damage, and Cell Proliferation Kit (BD Pharmingen). A 24-hour treatment with 250 nM of topotecan was used as a positive control. Data were acquired and analyzed using the FACSCalibur flow cytometer and the WinMDI 2.9 software respectively. Based on sytox^®green fluorescent intensity, cells with less than 2N DNA were excluded from analysis of γH2AX expression.