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Top: Hela cells were seeded overnight at 15,000 cells/90uL/well in a Costar black wall/clear bottom 96-well plate. The cells were untreated (control) or treated with1 mM H2O2 or 100 uM tert-butyl hydroperoxide (TBHP) for 30 min at 37°C. The ROS 570 assay solution (100uL/well) was added and incubated in a 5% CO2, 37°C incubator for 1 hour. The fluorescence signal were monitored at Ex/Em = 540/570 nm (cut off = 550 nm) with bottom read mode using FlexStation (Molecular Devices).
Bottom: Images of Hela cells stained with the Intracellular Total ROS Activity Assay Kit (Orange Fluorescence) (Cat # KA4075) in a Costar black wall/clear bottom 96-well plate. A: Untreated control cells. B: Cells treated with 100 uM tert-butyl hydroperoxide (TBHP) for 30min before staining.