Bassøe C F, Smith I, Sørnes S, Halstensen A, Lehmann A K
Division of Hematology, Haukeland University Hospital, Bergen, N-5021, Norway.
Methods. 2000 Jul;21(3):203-20. doi: 10.1006/meth.2000.1001.
The current study aims to review flow cytometric (FCM) parameters for the quantification of phagocytosis. A limitation of existing methods is their difficulty with accurate quantification of the phagocytic index, i.e., number of beads per phagocyte, in individual cell lines in mixed cell suspensions. We have quantified phagocytosis and the oxidative burst simultaneously using fluorescent beads coated with meningococcal outer membrane vesicles (OMV beads) by the conversion of dihydrorhodamine 123 (DHR-123) to rhodamine 123 (R-123). Both these processes depend on specific serum opsonins. After the incubation, staining with a fluorescent anti-CD14 monoclonal antibody succeeded in discriminating phagocytosing monocytes from neutrophils. The spectral overlaps between OMV beads, R-123, and anti-CD14 could be completely compensated. Percentage of phagocytosis and the phagocytic index were similar in monocytes and neutrophils, but the oxidative burst behaved differently. Two monocyte subpopulations were observed. Both subpopulations spontaneously converted some DHR-123 into R-123, whereas the reaction was triggered by phagocytosis in neutrophils. The total oxidative response increased with increasing phagocytic index in both cell types, but the oxidative burst in monocytes was about twice that of neutrophils. The oxidative ratio (mean R-123 fluorescence value divided by the phagocytic index) declined with time in monocytes, but increased in neutrophils. Our results demonstrate the need for careful attention to technical details. This single-laser, three-color FCM method facilitates the comparative research of phagocytosis and the oxidative burst in monocytes and neutrophils and provides a basis for a number of applications in hematology, infectious medicine, and immunology.
本研究旨在回顾用于吞噬作用定量分析的流式细胞术（FCM）参数。现有方法的一个局限性在于，它们难以准确量化混合细胞悬液中单个细胞系的吞噬指数，即每个吞噬细胞吞噬的珠子数量。我们通过将二氢罗丹明123（DHR-123）转化为罗丹明123（R-123），使用包被有脑膜炎球菌外膜囊泡的荧光珠子（OMV珠子）同时定量吞噬作用和氧化爆发。这两个过程均依赖于特定的血清调理素。孵育后，用荧光抗CD14单克隆抗体染色成功区分了吞噬的单核细胞和中性粒细胞。OMV珠子、R-123和抗CD14之间的光谱重叠可得到完全补偿。单核细胞和中性粒细胞的吞噬百分比和吞噬指数相似，但氧化爆发表现不同。观察到两个单核细胞亚群。两个亚群均能自发地将一些DHR-123转化为R-123，而在中性粒细胞中，该反应由吞噬作用触发。两种细胞类型中，总的氧化反应均随吞噬指数的增加而增加，但单核细胞的氧化爆发约为中性粒细胞的两倍。单核细胞的氧化比率（平均R-123荧光值除以吞噬指数）随时间下降，而中性粒细胞的氧化比率则上升。我们的结果表明需要仔细关注技术细节。这种单激光三色FCM方法有助于对单核细胞和中性粒细胞的吞噬作用和氧化爆发进行比较研究，并为血液学、传染病学和免疫学的多种应用提供了基础。
