Assay of Intracellular Hydrogen Peroxide Generation in Activated Cytometry Individual Neutrophils by Flow

Stimulation of neutrophils induces oxidative burst. The generations of oxygen radicals play a central role in host defense against bacteria. Once neutrophils are stimulated by inflammatory or bacterial stimuli, they produce superoxide anion in order to kill phagocytosed microorganisms. In the process, superoxide anion is converted to hydrogen peroxide (H
2
O
2
) by superoxide dismutase (EC 1.15.1.1) and can then be metabolized through the Fenton reaction to produce highly energetic hydroxyl radicals. Reactive oxygen species (ROS) are released into the phagosome and into the extracellular environment. Superoxide anion released outside cytoplasmic membrane determined by measuring reduction of cytochrome c (
1
). Although it provides the most specific assay to superoxide, it includes purification of neutrophils, needs at least 10
5
neutrophils per assay, and can only measure superoxide released outside neutrophils. A sensitive and simple method to measure ROS produced inside neutrophils is of particular interest. Flow cytometric assay may solve these problems. It provides a sensitive assay to measure intracellular ROS in individual neutrophils (
2
,
3
).