Electrochemistry and chemiluminescence techniques compared in the detection of NADPH oxidase activity in phagocyte cells |
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Authors: | A. Ashkenazi R.S. Marks |
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Affiliation: | a Department of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel b National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel c ILSE Katz Center for Meso and Nanoscale Science and Technology, P.O. Box 653, Beer-Sheva 84105, Israel |
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Abstract: | Several methodologies have been used in clinical chemistry for real-time assessment of NADPH oxidase primary product superoxide anion which dismutases to hydrogen peroxide. Among these methodologies, isoluminol chemiluminescence (CL) is considered to be one of the more sensitive and reliable techniques for the assessment of NADPH oxidase activity in neutrophils. The electrochemical technique was recently designed and also applied for real-time detection of NADPH oxidase activity in neutrophils but its reliability and sensitivity has not been investigated so far. In this study, isoluminol CL and electrochemical techniques were investigated and compared by monitoring the generation of superoxide and hydrogen peroxide in both PLB 985 cell line differentiated into neutrophil-like cells and human neutrophils. The electrochemical technique was shown to be as sensitive as that of CL and able to detect the reactive oxygen species (ROS) release of as low as 500 cells. Thus, the electrochemical technique could be used as an alternative to optical techniques for the evaluation of extracellular ROS in phagocyte cells. |
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Keywords: | CL, chemiluminescence DMSO, dimethyl sulfoxide DPI, diphenyleneiodonium chloride FACS, fluorescence-activated cell sorter MPO, myeloperoxidase HRP, horseradish peroxidase KRP, Krebs Ringer phosphate buffer PBS, phosphate-buffered saline PH, peak height PMA, phorbol myristate acetate PT, peak time ROS, reactive oxygen species SOD, superoxide dismutase |
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