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，我们考察了用流式细胞术检测大鼠外周血中中性粒细胞中活性氧的基本测量条件。用异硫氰酸荧光素(FITC)标记金黄色葡萄球菌作为吞噬颗粒，用流式细胞仪测定其吞噬活性。我们根据荧光强度确定了FITC的最佳浓度。对于活性氧产生活性的测定，采用了与超氧化物发生反应的氢乙啶(HE)，根据荧光强度的依赖关系确定了合适的浓度。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，还对相同的样品进行了硝基蓝四氮唑(NBT)还原。结果，两种测量方法之间确认了很强的相关性。接下来，We…更深入地研究了人外周血的基本测量条件。发现细胞反应后的溶血程序会影响中性粒细胞、淋巴细胞和单核细胞的流式细胞仪分布，并对手术方法进行了改进。另一方面，分离的中性粒细胞很快就会失去活性，从全血中分离后不久就有必要测量活性氧的产生。并与双氢罗丹明(DHR-123)、二氢荧光素二乙酸酯(DCFH-DA)进行比较，对高灵敏度的检测探针进行了检测。与DCFH-DA相比，DHR-123通过与髓过氧化物酶抑制剂NaN3共存而具有更高的敏感性。对于吞噬颗粒，采用了FITC标记的金黄色葡萄球菌和酵母多糖，但是病原体的培养是危险的，不容易用FITC标记。此外，酵母多糖可引起中性粒细胞聚集，不适合用于流式细胞术的检测。由于这些原因，使用了直径为1μm的带有荧光染料的羧酸盐微球，因此可以不需要这样的困难就可以测量吞噬活性。我们可以在不受HE和DHR-123干扰的情况下建立羧酸盐微球的检测波长，使吞噬和产生活性氧的同时分析成为可能；此外，利用流式细胞术的原理，通过改变检测波长可以同时测量多个指标，使得利用人血浆样本同时测量各种分子成为可能。常规的酶联免疫吸附试验(EL ISA)需要较大的样本量和较长的测定时间。另一方面，该方法具有较大的测量范围，但灵敏度低于ELISA法。综上所述，流式细胞术使我们能够测量多通道参数，这是我们直到最近才能做到的。较少

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