Measurement of Respiratory Burst Products, Released or Retained, During Activation of Professional Phagocytes
Measurement of Respiratory Burst Products, Released or Retained, During Activation of Professional Phagocytes
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DOI：
10.1007/978-1-62703-845-4_21
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发表时间：
2014-01-01
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影响因子：
--
通讯作者：
Dahlgren, Claes
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文献类型：
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作者：
Bylund, Johan;Bjornsdottir, Halla;Dahlgren, Claes
专业吞噬细胞是我们先天免疫系统的强效微生物杀手，它的激活与细胞对分子氧(O-2)的消耗增加有关。消耗的O-2被NADPH-氧化酶利用，通过单电子还原生成高活性氧物种(ROS)，最初生成超氧阴离子(O-2(-))，然后歧化为过氧化氢(H_2O_2)。ROS是强烈的杀菌分子，但也可能导致组织破坏，并能够驱动先天免疫系统和获得性免疫系统的免疫活性细胞进入凋亡。发展测量/量化呼吸爆发过程中吞噬细胞产生ROS的基本技术是非常重要的，为此已经使用了大量的方法。本章详细介绍了各种方法的选择，包括鲁米诺或异鲁米诺放大的化学发光，细胞色素c还原后吸光度的变化，以及PHPA氧化时荧光增强，特别强调如何区分细胞外释放的ROS和保留在细胞内细胞器内的ROS。这些技术在从基础吞噬细胞生物学到更面向临床的先天免疫机制和炎症研究中可以成为有价值的工具。
Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O-2). The consumed O-2 is utilized by an NADPH-oxidase to generate highly reactive oxygen species (ROS) by a one electron reduction, initially generating superoxide anion (O-2(-)) that then dismutates to hydrogen peroxide (H2O2). The ROS are strongly bactericidal molecules but may also cause tissue destruction, and are capable of driving immune competent cells of both the innate and the adaptive immune systems into apoptosis. The development of basic techniques to measure/quantify ROS generation by phagocytes during activation of the respiratory burst is of great importance, and a large number of methods have been used for this purpose. A selection of methods, including chemiluminescence amplified by luminol or isoluminol, the absorbance change following reduction of cytochrome c, and the fluorescence increase upon oxidation of PHPA, are described in detail in this chapter with special emphasis on how to distinguish between ROS that are released extracellularly, and those that are retained within intracellular organelles. These techniques can be valuable tools in research spanning from basic phagocyte biology to more clinically oriented research on innate immune mechanisms and inflammation.