Flow cytometer cell sorter

Cell cycle analysis was performed using a flow cytometer, cell sorter, and cell preparation system (Model no: A00-1-1102; Beckman Coulter, Indianapolis, IN, USA) and the ModFit LT 5.0 software package (Verity Software House, Topsham, ME, USA) was used for analysis. After drug exposure for 24, 48 and 72 h, the cells were collected fixed with 70% ice-cold ethanol and incubated for 30 min on ice. Then, cells were incubated with 10 mg/ml of propidium iodide and 5 mg/ml of ribonuclease-A in PBS for 45 min at 37 °C. ROS and 8-oxo-2'-deoxyguanosine levels were measured as described previously 24 (link). The doublet was excluded manually (Figure S2) and cell cycle gating was performed by auto analysis function of the software.
Flow cytometric determination of apoptosis by Alexa Flour 488 Annexin V/Dead cell apoptosis kit (Thermo Fischer, Oregon, USA) was performed using BD FACS Verse^TM Flow Cytometer (Cat. No: 651153). After drug exposure for 24, 48 and 72 h, the cells (1×10⁶ cells/ml) were collected in ice-cold PBS buffer and incubated with Alexa Flour 488 Annexin V and propidium iodide for 15 min and fluorescence emission at 530 nm and 575 nm was measured by flow cytometer cell sorter (Beckman Coulter) as described manufacturer. Data were analyzed and illustrated by BD FACSuite software version 1.0. Double discrimination was shown in supplemental material (Figure S2).