ROS measurement through DCF flow cytometry

To measure changes in ROS levels, molecular probe DCFDA (2’, 7’ – dichlorofluorescin diacetate) was used. After exposing parallel sets of SH-SY5Y ODD-Luc cells to either normoxia or hypoxia for 4 hr, media was removed, cells were washed with 1xPBS once, and then 1xPBS with 20 μM DCFDA was added in each well. Additional parallel sets of cells from both conditions were also treated with 5 mM H₂O₂ at the same time and were used as positive controls. Plates from both normoxia and hypoxia were then incubated at room temperature for 30 min in normoxic condition. DCFDA diffuses through the cell membrane and is deacetylated by intracellular esterases to a non-fluorescent form which is later oxidized by ROS into 2’, 7’ – dichlorofluorescein (DCF) which is a highly fluorescent compound. After incubation, fluorescence was measured through flow cytometry at the wavelength of excitation, 485 nm and emission, 535 nm, respectively. Production of ROS was measured as mean fluorescence index multiplied by respective cell counts and was expressed as fold change with respect to control.