Detection of intracellular ROS following BT treatment in ovarian cancer cell lines. (A) Detection of ROS by flow cytometry. Cells (1 × 106) were treated with 50 μM or 100 μM BT for 24 hrs. After treatment, cells were collected, washed and incubated with 5 μM C400 and analyzed by flow cytometry. Data was presented as relative-fluorescence intensity in a 2-dimensional FACS profile (standardized gating, 20,000 events). Enhanced ROS generation was shown by shift in peaks in treated cells as compared to control (labeled as ‘C’). All experiments were performed in triplicate. (B) Effect of antioxidant ascorbic acid on BT treated cells. Cells were pre-treated with 1 mM ascorbic acid for 2 hrs before addition of drug and further incubated for 48 hrs with both BT and ascorbic acid. Cell viability was determined by PrestoBlue reagent. Control (untreated) cells were considered as 100% viable against which treated cells were compared. The results represent % viability recovery when compared with 100 μM BT treated cells. Experiments were performed in triplicate. Data was expressed as mean ± SD of triplicate experiments. *p < 0.05 and **p < 0.01, as compared to control, Students’ t test.