Flow Cytometric Detection of Reactive Oxygen Species

Reactive oxygen species (ROS) are molecules containing hydroxyl radicals or peroxides with unpaired electrons. In healthy aerobic cells, ROS are produced naturally as a byproduct of oxidative phosphorylation, oxidoreductase enzymes, or metal catalyzed oxidation at a controlled rate. However, ROS can be induced under some stress conditions especially exposure to environmental oxidants and certain drugs that leads to oxidative stress. Exceed ROS can cause damages in the building blocks of cells including DNA, proteins, and lipids, and eventually results in cell death. Cell-permeant 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is a widely used ROS indicator. The reduced non-fluorescent fluorescein H2DCFDA can be oxidized and converted into fluorescent 2’, 7’-dichlorofluorescein (DCF) by intracellular ROS. In this protocol, we applied H2DCFDA to label the intracellular ROS and detected the DCF intensity by flow cytometry.