Measurement of Respiratory Burst Products, Released or Retained, During Activation of Professional Phagocytes

Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increased cellular consumption of molecular oxygen (O2). The O2 molecules consumed are reduced by electrons delivered by a membrane localized NADPH-oxidase that initially generate one- and two electron reduced superoxide anions (O2 -) and hydrogen peroxide (H2O2), respectively. These oxidants can then be processed into other highly reactive oxygen species (ROS) that can kill microbes, but that may also cause tissue destruction and drive other immune cells into apoptosis. The development of basic techniques to measure and quantify ROS generation by phagocytes is of great importance, and a large number of methods have been used for this purpose. A selection of methods (including chemiluminescence amplified by luminol or isoluminol, absorbance change following reduction of cytochrome c, and fluorescence increase upon oxidation of PHPA) are described in detail in this chapter with special emphasis on how to distinguish between ROS that are released extracellularly, and those that are retained within intracellular organelles. These techniques can be valuable tools in research spanning from basic phagocyte biology to diagnosis of diseases linked to the NADPH-oxidase and more clinically oriented research on innate immune mechanisms and inflammation.