Determination of ROS generation by flow cytometry and fluorescent microscopy

Dihydroethidium (DHE) (Invitrogen, Carlsbad, CA) assay is used to determine intracellular O₂⁻ levels. BM-EPCs were incubated with DHE (10⁻⁶ mol/L) for 30 min in a cell incubator. The fluorescence intensity for 10,000 events was measured using FACS (fluorescence activated cell sorting, BD). Culture plates were read at 518/605 nm in a SpectraMax M2e microplate reader (Molecular Devices, Sunnyvale, CA) and images were captured under the Olympus IX71 fluorescence microscope (Olympus, Japan).