Superoxide measurement system by pH-jump method

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For direct measurement of the ESR signal of superoxide anion (O^-_) produced in biological. O^-_generated at a physiological pH was trapped in alkaline media instead of by a rapid freezing method, and then its signal was measured by ESR spectroscopy at 77K.A reaction mixture for O^-_generation, such as neutrophils and NADPH oxidase in neutrophils, was incubated at a physiological pH (pH7.0-7.5) for a suitable reaction period (30 s), then an aliquot (300 mul) was pipetted out and squirted into 600 mul of 0.5 M NaOH to stabilize O^-_(pH-jump). The alkaline mixture was promptly introduced into an ESR tube and frozen by dipping the tube directly into a cooling liquid. A typical signal of O^-_ was detected by ESR spectroscopy and the amount of trapped O^-_ was measured quantitatively at 77 K.Based on this pH-jump method, we developed the superoxide measuring system which is controlled by the personal computer.This system should be widely used for direct measurement of O^-_ produced in biological system at physiological pH.