4.5. Measurement of ROS Production

Di-chloro-dihydro-fluorescein diacetate assay was used to measure ROS production in MCF7 and FaDu cells after treatment with HAL, PLEC and MRC for 12, 24 and 48 h. The DCFH-DA crosses the cell membrane and enzymatically hydrolyzed by intracellular eterases to non-fluorescent di-chloro-dihydro-fluorescin (DCFH), which is then oxidized to highly fluorescent dichloro-fluorescin (DCF) in the presence of intracellular ROS. Cells were seeded in 96-well black plates at a density of 1×10⁵ cells/well in 50 µL culture medium and allowed to adhere overnight in a CO₂ incubator at 37 °C. The next day, the cells were incubated for 45 min at 37 °C in presence of 5 µM of DCFH-DA, washed in HBSS and then treated with indicated concentrations of HAL, PLEC and MRC for 12, 24 and 48 h. The readings were taken at 485/20 nm excitation and 528/20 nm emission.