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Detection of ROS in Hela cells. Hela cells were seeded overnight at 15000 cells/90 uL/well in a Costar black wall/clear bottom 96-well plate. The cells were treated (1 mM H2O2 or 100 uM tert-butyl hydroperoxide) or untreated (control) for 30 min at 37°C. The ROS Deep Red stain solution (100 uL/well) was added and incubated in a 5% CO2, 37°C incubator for 1 hour. The fluorescence signal was monitored at Ex/Em = 650/675 nm (cut off = 665 nm) with bottom read mode using FlexStation.