Analysis and assessment of the capacity of neutrophils to produce reactive oxygen species by application of flow cytometry.

首先，为了推进实验动物的各项研究，我们探讨了利用流式细胞术检测大鼠外周血中性粒细胞活性氧的基本测量条件。为了用流式细胞术检测金黄色葡萄球菌的吞噬活性，用异硫辛酸（FITC）荧光标记金黄色葡萄球菌，并将其作为吞噬颗粒。我们根据荧光强度确定了FITC的最佳浓度。测定反应产氧活性时，采用与超氧化物反应的氢乙胺（HE），通过荧光强度的依赖关系确定合适的浓度。此外，通过检测不同波长的FITC和HE，可以同时测量这两种活性。此外，还对同一样品进行了硝基蓝四氮唑（NBT）还原法。结果证实了两种测量方法之间的强相关性。接下来，我们进一步研究了人体外周血的基本测量条件。发现细胞反应后的溶血操作会影响中性粒细胞、淋巴细胞和单核细胞的流式细胞仪分布，我们改进了操作方法。另一方面，分离的中性粒细胞很快失去活性，因此有必要在与全血分离后不久测量活性氧的产生。然后将HE与二氢膦丹明(DHR) -123、二氯荧光素二乙酸酯（DCFH-DA）进行比较，检验其高灵敏度。与DCFH-DA相比，DHR-123因与髓过氧化物酶抑制剂NaN3共存而具有更高的敏感性。吞噬颗粒采用FITC标记金黄色葡萄球菌和酵素，但用FITC标记病原菌培养危险且不易处理。此外，zymosan引起中性粒细胞聚集，不适合流式细胞术的测量。因此，使用直径为1 μ m的荧光染料羧酸盐微球，可以省去这些困难来测量吞噬活性。建立了不受HE和DHR-123干扰的羧酸盐微球检测波长，实现了对吞噬和活性氧产生的同时分析。此外，通过应用流式细胞术的原理，可以通过改变检测波长来同时测量多个指标，从而可以使用人血浆样品同时测量各种分子。传统的酶联免疫吸附法（ELISA）需要更大的样本量和时间来测量。另一方面，该方法的测定范围较大，但灵敏度较ELISA法差。综上所述，流式细胞术方法使我们能够测量多通道参数，这是我们直到最近才能够做到的。少

First, in order to advance the various researches in laboratory animals, we examined fundamental measurement conditions for detecting reactive oxygen species of rat peripheral neutrophils by use of flow cytometry. For measuring phagocytic activity with flow cytometry, Staphylococcus aureus was labeled with fluoresce in isothiocynate (FITC) and used as phagocytic particles. We decided the optimal concentrations of FITC in regard to the fluorescent intensity. As for the measurement of reactive oxygen-producing activity, hydroethidine (HE) which reacts with superoxide was used and the suitable concentration was determined by dependency of the fluorescent intensity. Furthermore, simultaneous measurement of both of these activities was made possible by detecting FITC and HE at the different wave length. In addition, nitroblue tetrazolium (NBT) reduction method was also performed using the same samples. As a result, strong correlations were confirmed between both measurement methods.Next, we … More investigated the fundamental measurement conditions in case of human peripheral blood. It was found that hemolysis procedure after the cellular reaction could affect flow cytometry distribution of neutrophils, lymphocytes and monocytes, and we improved the operation methods. On the other hand, isolated neutrophils lose activity soon, and there was a necessity to measure reactive oxygen production soon after the separation from the whole blood. Then we tried to examine the high sensitivity detection probe by comparing HE with Dihydrorhodamine (DHR) -123, Dichrolo Fluorescein Diacetate (DCFH-DA). DHR-123 was superior in sensitivity, by coexisting NaN3 which is the inhibitor of myeloperoxidase, compared with DCFH-DA. Concerning the phagocytic particles, FITC-labeled Staphylococcus aureus and zymosan were used, but the culture of pathogens is dangerous and not easy to handle to label with FITC. In addition, zymosan caused aggregation of neutrophils and was not suitable for the measurement of flow cytometry method. For those reasons, Carboxylate Microspheres of the diameter 1 μ m with fluorescence dye was used, and it became possible to measure phagocytic activity without such difficulties. We could set up the detection wave length of Carboxylate Microspheres without interference of those of HE and DHR-123, and simultaneous analyses of phagocytosis and reactive oxygen production became possible.Furthermore, by applying the principle of flow cytometry that can measure several indices simultaneously by changing detection wave length, the simultaneous measurement of various molecules using human plasma samples became possible. Conventional enzyme-linked immunosorbent assay (ELISA) takes larger sample volume and takes time for the measurement. On the other hand, this method have greater range of measurement, but the sensitivity was poorer than ELISA method. Taken together, flow cytometry method is enable us to measure multi-channel parameters that we could not do until recently. Less