ABSTRACT
This procedure has been developed to monitor the production of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anions, in granulocytes. The capacity to produce ROS, which is called the oxidative burst, is critical for the degradation of material by granulocytes. The oxidative burst is also implicated in inflammatory damage to tissues. In this assay, cells are exposed to a fluorescent probe to assess the production of intracellular ROS. Oxidation of the probe to a fluorescent dye, such as dichlorofluorescin diacetate (DCFH-DA) or dihydrorhodamine 123 (DHR 123) has been used to detect ROS formation. After cellular incorporation of the probe, the cells are exposed to an activator, which stimulates the production of ROS. The fluorescence of the probe, at a time period, reveals the relative amount of intracellular ROS in a cell population. Finally, the oxidative burst is evaluated on a kinetic time basis using flow cytometry. The results should demonstrate that the production of ROS increases with time. One way to demonstrate the time effect is to make a control sample containing no activator.
