Measurement of Respiratory Burst Products Generated by Professional Phagocytes

Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O₂ ). The burst of O₂ consumption is utilized by an NADPH-oxidase to generate highly-reactive oxygen species (ROS) starting with one and two electron reductions to generate superoxide anion (O₂ ⁻ ) and hydrogen peroxide (H₂ O₂ ), respectively. ROS are strongly bactericidal but may also cause tissue destruction and induce apoptosis in other immune competent cells of both the innate and the adaptive immune systems. Thus, the development of basic techniques to measure/quantify ROS generation/release by phagocytes during activation of the respiratory burst is of great importance, and a large number of techniques have been used for this purpose. Three of these techniques, chemiluminescence amplified by luminol/ isoluminol, the absorbance change following reduction of cytochrome c, and the fluorescence increase upon oxidation of p-hydroxyphenylacetate, are described in detail in this chapter. These techniques can be valuable tools in research spanning from basic phagocyte biology to more clinically-oriented research on innate immune mechanisms and inflammation.