Analysis and assessment of the capacity of neutrophils to produce reactive oxygen species in a 96-well microplate format using lucigenin- and luminol-dependent chemiluminescence
Analysis and assessment of the capacity of neutrophils to produce reactive oxygen species in a 96-well microplate format using lucigenin- and luminol-dependent chemiluminescence
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DOI：
10.1016/s0022-1759(97)00159-2
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发表时间：
1997-12-15
影响因子：
2.2
通讯作者：
Sugawara, K
中科院分区：
文献类型：
--
作者：
Hasegawa, H;Suzuki, K;Sugawara, K
化学发光（CL）法已被用于测定吞噬细胞产生活性氧（ROS）的能力。为了获得灵敏度更高的多通道微孔板光子计数CL分析仪的最佳测量条件，我们研究了影响lucigenin依赖性CL （LgCL）测量人类中性粒细胞变异性的因素；用调理酶（OZ）或肉豆蔻酸酯（PMA）刺激。我们使用50 μ l中性粒细胞（3 × 10(6)个细胞/ml）和50 μ l 0.5 mM lucigenin（每孔），以及100 μ l OZ (5 mg/ml)(当zymosan与10-20%的血清或；100 μ l PMA溶液（1 × 10(-6) M），在37℃的连续测量中自动定时混合和检测。此外，我们使用超氧化物歧化酶（SOD）、过氧化氢酶、去铁胺和氮化钠（NaN3）等ROS代谢调节剂研究了各种ROS对LgCL和鲁米诺依赖性CL （LmCL）的贡献。LmCL可被超氧化物歧化酶（SOD）抑制，但不受其他药物的抑制，而LmCL可被NaN和去铁胺抑制。因此，LmCL检测超氧阴离子具有高选择性，而LmCL检测髓过氧化物酶（MPO）介导的次氯酸形成，这种基于微球板的多重测量有助于准确评估吞噬功能。(C) 1997爱思唯尔科学有限公司
The chemiluminescence (CL) assay has been used to measure the reactive oxygen species (ROS)-generating capacity of phagocytes. To achieve more optimal measurement conditions for a multi-channel microplate photon-counting CL analyzer with the cooled charge-coupled device (CCD) camera which offers enhanced sensitivity, we investigated factors affecting the variability in lucigenin-dependent CL (LgCL) measurement of human neutrophil; stimulated with either opsonized zymosan (OZ) or phorbol myristate acetate (PMA). We obtained sensitive LgCL responses with good reproducibility and rapid data-acquisition using 50 mu l neutrophils (3 x 10(6) cells/ml) and 50 mu l of 0.5 mM lucigenin per well, in addition to either 100 mu l of OZ (5 mg/ml) when zymosan was opsonized with 10-20% serum or; 100 mu l of PMA solution (1 x 10(-6) M) with automatic regular intervals of mixing and detection during the continuous measurement at 37 degrees C. Furthermore, we studied the contribution of various ROS to LgCL and luminol-dependent CL (LmCL) using modulators of ROS metabolism including superoxide dismutase (SOD), catalase, deferoxamine and sodium azide (NaN3). LgCL was inhibited by SOD but not by the other agents, whereas LmCL was inhibited by NaN, and deferoxamine, Thus, it was demonstrated that LgCL detects the superoxide anion with high selectivity whereas the LmCL assay measures myeloperoxidase (MPO)-mediated formation of hypochlorous acid, Such microplate-based multiple measurements facilitate the accurate assessment of phagocytic function. (C) 1997 Elsevier Science B.V.