Intracellular Total ROS Activity Assay

1. Prepare samples and controls., 2. Dilute 10X Cellular Assay Buffer 1:10 with diH2O., 3. Reconstitute FAM-FLICA with 50 µL DMSO., 4. Dilute FAM-FLICA 1:5 by adding 200 µL PBS., 5. Add diluted FAM-FLICA to each sample at 1:30-1:60 (e.g., spike at 1:30 by adding 10 µL to 290 µL sample)., 6. Incubate approximately 1 hour., 7. Remove media and wash cells 3 times: add 1X Cellular Wash Buffer and spin cells., 8. If desired, label with additional stains, such as Hoechst 33342, Propidium Iodide, 7-AAD, or an antibody., 9. If desired, fix cells., 10. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM-FLICA excited at 492 nm and emits at 520 nm.