Intracellular ROS Levels Under Stress

Intracellular ROS levels under normal and stress conditions were detected using a 2′,7′–DCFDA assay kit. The A2780/CP and A2780 cells were treated with 1 µg/mL PEI and 2.5 µg/mL CIS, as well as with PM/C and PMC complexes (at concentrations of 1 µg/mL PEI, 1 µg/mL MNP, and 2.5 µg/mL of CIS), compared to untreated cells, in the presence and absence of SMF for 48 h, which were prepared in RPMI medium supplemented with 10% FBS in 6-well cell culture plates (SPL Life Sciences Co., Ltd. Korea). After treatment, the cells were prepared immediately, according to the manufacturer’s instructions. Briefly, the cells were washed with PBS. The samples were then suspended in a conical test tube with 20 µM DCFDA in 1X buffer and incubated at 37°C in the dark for 30–45 min. ROS production was monitored immediately using a FACSCalibur Becton-Dickinson flow cytometer (Franklin Lakes, NJ). DCFDA flow cytometric data were analyzed using FlowJo software (version 7.6.1) (22 (link), 27 (link), 45 (link)).