Annexin 5 fitc ann

HCEnC-21T cells were seeded in a 6-well plate and cultured for 24 h. Cells were treated with/without MN (50 μM) or 4-OHE₂ (10 μM), and incubated for 16 h in estrogen-free medium. Cells were then trypsinized, washed in cold PBS, and incubated in the dark with 3 μL of Annexin-V/FITC (Ann) (Thermo Fisher Scientific) and 2 μL of propidium iodide (PI, Sigma-Aldrich) in a total volume of 50 μL for 30 min at room temperature. Cells were re-suspended in 2 mL of Isoflow sheath fluid (Beckman Coulter, Fullerton, CA) and 20,000 live cells were acquired on a LSR II (BD Biosciences) with DIVA software (BD Biosciences) gated for PI and Annexin V with unstained cells as a negative control. Data were then analyzed with Summit 4.3 (Beckman Coulter, Fullerton, CA).

HCEnC-21T control (NQO1^+/+) and NQO1^−/− cells were seeded in a 6-well plate and cultured for 24 hours prior to treating with 50μM MN in low-glucose DMEM) and incubated for 5 hours. Cells were then trypsinized, washed in cold PBS, and incubated in the dark with 3μl of Annexin-V/FITC (Ann) (Thermo Fisher, Waltham, MA) and 2μl of PI (Sigma-Aldrich) in a total volume of 50μl for 30 minutes at room temperature. Cells were re-suspended in 2ml of Isoflow sheath fluid (Beckman Coulter, Fullerton, CA) and 20,000 live cells were acquired on a LSR II (BD Biosciences) with DIVA software (BD Biosciences) gated for PI and Annexin V with unstained cells as negative control. Data were then analyzed with Summit 4.3 (Beckman Coulter, Fullerton, CA).