Direct measurement of free chloride concentrations in the phagolysosomes of human neutrophils

Analytical Chemistry
Richard G Painter, Guoshun Wang

Abstract

Human neutrophils synthesize hypochlorous acid, a nonradical oxidant, as one of the antimicrobial agents to kill phagocytosed pathogens within phagolysosomes. The production of HOCl is catalyzed by myeloperoxidase using chloride anions (Cl-). Even though various approaches have been documented to measure cytosolic Cl- concentrations, direct detection of phagolysosomal free Cl- in a real-time fashion is not available. Here we report the development of a new dual-labeled fluorescent probe by conjugation of the chloride-sensitive 6-methoxyquinoline and chloride-insensitive tetramethylrhodamine to the zymosan particles. On the surface of the zymosan particles, the chloride-sensitive fluorophore responded linearly to chloride concentrations when plotted according to the Stern-Volmer collisional quenching equation and the chloride-insensitive fluorophore served as an internal reference. After phagocytosis into neutrophils, the probe was able to monitor intraphagosomal chloride levels authentically. Human neutrophils in the medium containing 122 mM chloride had a phagolysosomal chloride level of 73.3 +/- 12.2 mM (N = 5). In contrast, the neutrophils in a chloride-free isoosmotic medium had a chloride level of 6.6 +/- 1.9 mM (N = 5) in...Continue Reading

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Citations

Jun 7, 2008·Chemical Communications : Chem Comm·Elizabeth Anne HillardChristian Amatore
Oct 7, 2009·Proceedings of the National Academy of Sciences of the United States of America·Deri MorganThomas E DeCoursey
Jan 12, 2011·Proceedings of the National Academy of Sciences of the United States of America·Andrea L RadtkeMary X O'Riordan
Aug 14, 2012·Antioxidants & Redox Signaling·Christine C Winterbourn, Anthony J Kettle
Jul 18, 2008·Philosophical Transactions. Series A, Mathematical, Physical, and Engineering Sciences·Marat MukhtarovPiotr Bregestovski
Mar 15, 2013·Journal of Innate Immunity·Yun ZhouGuoshun Wang
Oct 16, 2012·Journal of Leukocyte Biology·Seymour J KlebanoffWilliam M Nauseef
Mar 21, 2008·Journal of Leukocyte Biology·Richard G PainterGuoshun Wang
Jan 22, 2010·Journal of Leukocyte Biology·Richard G PainterGuoshun Wang
Dec 14, 2011·PloS One·Lorenzo AlbertazziFabio Beltram
Oct 18, 2011·Free Radical Biology & Medicine·Anthony J KettleRobert F Anderson
Sep 28, 2010·Free Radical Biology & Medicine·Ryan W BonvillainGuoshun Wang
Feb 19, 2008·Journal of Neuroscience Methods·Olga MarkovaPiotr Bregestovski
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Oct 24, 2009·Archives of Biochemistry and Biophysics·Christine SuquetJames K Hurst

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