| A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells. | |
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MedLine Citation:
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PMID: 16450867 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Conventionally, a semi-quantitative microscopic nitroblue tetrazolium (NBT) assay is used to determine the production of superoxide anion (O2(-)) in various phagocytic cells. This microscopic assay is conducted by counting the cells containing blue NBT formazan deposits, which are formed by reduction of the membrane permeable, water-soluble, yellow-colored, nitroblue tetrazolium (Y-NBT) by O2(-). However, this assay is semi-quantitative and is prone to observer bias. In the present study, we modified the NBT assay by dissolving the blue formazan particles using 2M potassium hydroxide and dimethylsulfoxide and then measured its absorbance using a microplate reader at 620nm. The absorbance of dissolved NBT increased in proportion to cell number (r = 0.9907), incubation time, and stimulus concentration. To test the usefulness of this modified assay, we compared the abilities of a number of types of phagocytic cells to produce O2(-). The cells examined included murine macrophage cell lines (RAW 264.7 and J774), freshly prepared murine peritoneal macrophages and neutrophils, a human myeloid cell line (PLB-985), and freshly prepared human peripheral blood neutrophils. In addition, we demonstrate that nitric oxide produced by RAW 264.7 cells does not interfere with the modified colorimetric NBT assay. Taken together, our results indicate that the modified colorimetric NBT assay is simple, sensitive, and quantitative, and that it can be used to determine the amounts of intracellular O2(-) produced by phagocytic cells. Thus, this assay is sensitive enough to measure, quantitatively, even the small amounts of O2(-) produced in monocytes and macrophages that are not detectable by the conventional microscopic NBT assay. |
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Authors:
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Hyung Sim Choi; Jun Woo Kim; Young-Nam Cha; Chaekyun Kim |
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Publication Detail:
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Type: Comparative Study; Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Journal of immunoassay & immunochemistry Volume: 27 ISSN: 1532-1819 ISO Abbreviation: J Immunoassay Immunochem Publication Date: 2006 |
Date Detail:
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Created Date: 2006-02-02 Completed Date: 2006-03-15 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 100963688 Medline TA: J Immunoassay Immunochem Country: United States |
Other Details:
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Languages: eng Pagination: 31-44 Citation Subset: IM |
Affiliation:
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Laboratory of Cell Signaling, Inha University College of Medicine, Incheon, Korea. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Biological Assay / methods Cell Line Cells, Cultured Cytochromes c / metabolism Dimethyl Sulfoxide Humans Hydroxides Macrophages / metabolism* Mice Mice, Inbred C57BL Neutrophils / metabolism* Nitric Oxide / metabolism Nitroblue Tetrazolium / metabolism* Oxidation-Reduction Potassium Compounds Superoxide Dismutase / antagonists & inhibitors Superoxides / metabolism* |
| Chemical | |
Reg. No./Substance:
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0/Hydroxides; 0/Potassium Compounds; 10102-43-9/Nitric Oxide; 11062-77-4/Superoxides; 1310-58-3/potassium hydroxide; 298-83-9/Nitroblue Tetrazolium; 67-68-5/Dimethyl Sulfoxide; 9007-43-6/Cytochromes c; EC 1.15.1.1/Superoxide Dismutase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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