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

首先，为了推进在实验动物中的各种研究，我们研究了使用流式细胞术检测大鼠外周中性粒细胞的活性氧的基本测量条件。为了用流式细胞术测量吞噬活性，将金黄色葡萄球菌用异硫氰酸酯中的荧光（FITC）标记并用作吞噬颗粒。我们根据荧光强度确定了FITC的最佳浓度。在活性氧产生活性的测定中，使用了与超氧化物反应的乙氢定（HE），并通过荧光强度的依赖性确定了合适的浓度。此外，通过在不同波长下检测FITC和HE，可以同时测量这两种活性。此外，还使用相同的样品进行了氮蓝四唑（NBT）还原法。因此，两种测量方法之间的强相关性得到证实。 关于我们 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程序对中性粒细胞、淋巴细胞和单核细胞的流式细胞仪分布有影响，并对操作方法进行了改进。另一方面，分离的中性粒细胞很快失去活性，并且有必要在从全血分离后不久测量活性氧的产生。然后通过HE与二氢罗丹明（DHR）-123、二氯罗丹明二乙酸酯（DCFH-DA）的比较，对高灵敏度的检测探针进行了尝试。与DCFH-DA相比，DHR-123在存在髓过氧化物酶抑制剂NaN 3的情况下灵敏度上级。对于吞噬颗粒，使用FITC标记的金黄色葡萄球菌和酵母聚糖，但病原体培养是危险的，并且不容易处理FITC标记。此外，酵母多糖引起中性粒细胞聚集，不适合流式细胞术方法的测量。由于这些原因，使用具有荧光染料的直径1 μ m的羧酸盐微球，并且可以在没有这种困难的情况下测量吞噬活性。我们可以设置羧酸盐微球的检测波长而不受HE和DHR-123的干扰，使吞噬和活性氧产生的同时分析成为可能，并应用流式细胞术通过改变检测波长可以同时测量多个指标的原理，使人血浆样品中多种分子的同时测量成为可能。传统的酶联免疫吸附测定法（ELISA）需要较大的样品体积和测量时间。另一方面，该方法具有更大的测量范围，但灵敏度比ELISA方法差。总之，流式细胞术方法使我们能够测量多通道参数，直到最近我们才能做到。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