A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells
A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells
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DOI：
10.1080/15321810500403722
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发表时间：
2006-01-01
影响因子：
--
通讯作者：
Kim, C
中科院分区：
文献类型：
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作者：
Choi, HS;Kim, JW;Kim, C
传统上，半定量微观硝基蓝四氮唑（NBT）测定用于测定各种吞噬细胞中超氧阴离子（O-2(-)）的产生。这种显微分析是通过计数含有蓝色NBT甲酸沉积物的细胞来进行的，这些沉积物是由O-2（-）还原膜渗透性，水溶性，黄色的硝基蓝四氮唑（Y-NBT）形成的。然而，这种分析是半定量的，容易产生观察者偏差。在本研究中，我们修改了NBT法，用2M氢氧化钾和二甲基亚砜溶解蓝色甲醛颗粒，然后使用微孔板阅读器在620nm处测量其吸光度。溶出的NBT吸光度与细胞数、孵育时间和刺激浓度成正比（r = 0.9907）。为了检验这种改进的检测方法的有效性，我们比较了几种类型的吞噬细胞产生O-2（-）的能力。所检测的细胞包括小鼠巨噬细胞系（RAW 264.7和J774）、新鲜制备的小鼠腹腔巨噬细胞和中性粒细胞、人骨髓细胞系（PLB-985）和新鲜制备的人外周血中性粒细胞。此外，我们证明了由RAW 264.7细胞产生的一氧化氮不会干扰改进的比色法NBT测定。综上所述，我们的结果表明，改进的比色法NBT测定方法简单、灵敏、定量，可用于测定吞噬细胞产生的细胞内O-2（-）的量。因此，这种检测方法足够灵敏，甚至可以定量测量单核细胞和巨噬细胞中产生的少量O-2(-)，而传统的显微镜下的NBT检测方法无法检测到。
Conventionally, a semi-quantitative microscopic nitroblue tetrazolium (NBT) assay is used to determine the production of superoxide anion (O-2(-)) in various phagocytic cells. This microscopic assay is conducted by counting the cells containing blue NBT formazan deposits, which are formed by reduction of he membrane permeable, water-soluble, yellow-colored, nitroblue tetrazolium (Y-NBT) by O-2(-). 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 O-2(-). 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 O-2(-) produced by phagocytic cells. Thus, this assay is sensitive enough to measure, quantitatively, even the small amounts of O-2(-) produced in monocytes and macrophages that are not detectable by the conventional microscopic NBT assay.