Identification of ROS using oxidized DCFDA and flow-cytometry.

Cells constantly generate reactive oxygen species (ROS) during aerobic metabolism. The ROS generation plays an important protective and functional role in the immune system. The cell is armed with a powerful antioxidant defense system to combat excessive production of ROS. Oxidative stress occurs in cells when the generation of ROS overwhelms the cells' natural antioxidant defenses. ROS and the oxidative damage are thought to play an important role in many human diseases including cancer, atherosclerosis, other neurodegenerative diseases and diabetes. Thus, establishing their precise role requires the ability to measure ROS accurately and the oxidative damage that they cause. There are many methods for measuring free radical production in cells. The most straightforward techniques use cell permeable fluorescent and chemiluminescent probes. 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) is one of the most widely used techniques for directly measuring the redox state of a cell. It has several advantages over other techniques developed. It is very easy to use, extremely sensitive to changes in the redox state of a cell, inexpensive and can be used to follow changes in ROS over time.