Facsdiva 7

The overproduction of intracellular ROS was measured with dihydroethidium (DHE) staining (D11347, ThermoFisher Scientific, Waltham, MA, USA). JJN3 and U266 cells were seeded at a density of 2 × 10⁵ cells/mL/well into 24-well plates and incubated for 24 h before treatments with 20–80 µg/mL of 5,6 α/β-ECs or with 250 or 500 μM H₂O₂ as positive controls. Cells were further incubated with 2 µM DHE for 15 min at 37 °C. The production of O²⁻ was monitored by oxidized DHE that exhibits an orange/red fluorescence [11 (link)]. The signal was collected from a minimum of 10⁴ events for each sample using the BD FACSCanto II flow cytometer (BD Biosciences) and data were processed with the BD FACSDiva 7 software (BD Biosciences).
JJN3 or U266 cells were plated for 24 h in 24-well plates (2 × 10⁵ cells/well), treated with 5,6 α-EC or 5,6 β-EC (20–80 µg/mL) alone or after a 2 h-pretreatment with 400 µM vitamin E (Vit E, #258024, Sigma-Aldrich, Saint Louis, MO, USA). After 24 or 48 h of treatment, cells were stained with 1 µg/mL PI (#P4170, Sigma-Aldrich, Saint Louis, MO, USA). At least, 10⁴ events per sample were acquired and analyzed by flow cytometry BD FACSCanto II. Data were processed using BD FACSDiva 7 software (BD Biosciences, Franklin Lakes, NJ, USA).