FLOW CYTOMETRIC ANALYSIS OF RESPIRATORY BURST ACTIVITY IN PHAGOCYTES WITH HYDROETHIDINE AND 2',7'-DICHLOROFLUORESCIN
FLOW CYTOMETRIC ANALYSIS OF RESPIRATORY BURST ACTIVITY IN PHAGOCYTES WITH HYDROETHIDINE AND 2',7'-DICHLOROFLUORESCIN
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DOI：
10.1002/jlb.47.5.440
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发表时间：
1990-05-01
影响因子：
5.5
通讯作者：
VALET, G
中科院分区：
文献类型：
--
作者：
ROTHE, G;VALET, G
使用氢乙啶 (HE) 和 2'',7''-二氯荧光素 (DCFH) 对白细胞中的活性氧代谢物进行流式细胞术测量。在无细胞比色皿测定中，氢乙啶和 DCFH 均被 H2O2 和过氧化物酶快速氧化为溴化乙锭 (EB) 和 2'',7''-二氯荧光素 (DCF)，但单独使用 H2O2 则不会，而只有 HE 被 KO2（O2- 来源）氧化。静止的淋巴细胞、单核细胞和中性粒细胞自发地将HE氧化为EB，而DCFH仅被低程度氧化。在佛波醇 12-肉豆蔻酸酯 13-乙酸酯诱导的呼吸爆发期间，中性粒细胞的 EB 红色荧光增加了 6.9 倍，DCF 绿色荧光增加了 12.5 倍，在大肠杆菌诱导的呼吸爆发期间，中性粒细胞的 EB 红色荧光增加了 6.9 倍，DCF 绿色荧光增加了 12.5 倍。与自发的 HE 氧化不同，呼吸爆发期间的 HE 或 DCFH 氧化不能被 10 mM NaNe 抑制，这表明呼吸爆发期间细胞氧化剂的非线粒体来源，例如产生 O2- 的 NADPH 氧化酶。在同时负载 HE 和 DCFH 的受刺激中性粒细胞中，DCFH 的氧化减少，但 HE 的氧化没有减少。由静息中性粒细胞与细胞外 H2O2 孵育诱导的细胞内 DCFH 氧化不受 HE 存在的影响。这表明HE在中性粒细胞活性氧代谢中比DCFH更早被氧化，即被早期氧代谢物如O2-氧化，而DCFH部分被H2O2和吞噬体过氧化物酶氧化。在同时细胞染色过程中，HE 和 DCFH 的差异氧化允许分析脓毒症患者中多达三个功能不同的中性粒细胞群。这对于确定静止和受刺激的白细胞中与疾病相关的氧代谢变化很有意义。
Hydroethidine (HE) and 2'',7''-dichlorofluorescin (DCFH) were used for the flow cytometric measurement of reactive oxygen metabolites in leukocytes. Hydroethidine and DCFH were both rapidly oxidized in a cell-free cuvette assay to ethidium bromide (EB) and 2'',7''-dichlorofluorescein (DCF) by H2O2 and peroxidase, but not by H2O2 alone, while only HE was oxidized by KO2, a source of O2-. Quiescent lymphocytes, monocytes, and neutrophils spontaneously oxidized HE to EB, while DCFH was only oxidized to a low degree. Neutrophils increased 6.9-fold in EB red fluorescence and 12.5-fold in DCF green fluorescence during the respiratory burst induced by phorbol 12-myristate 13-acetate or 6.1-fold and 4.7-fold, respectively, during the respiratory burst induced by Escherichia coli bacteria. The HE or DCFH oxidation during the respiratory burst, unlike the spontaneous HE oxidation, was not inhibitable by 10 mM NaNe indicating a non-mitochondrial source of cellular oxidants during the respiratory burst such as NADPH oxidase, which produces O2-. The oxidation of DCFH, but not of HE, was decreased in stimulated neutrophils, which were simultaneously loaded with HE and DCFH. Intracellular DCFH oxidation induced by incubation of resting neutrophils with extracellular H2O2 was not influenced by the presence of HE. This indicates that HE is oxidized at an earlier step in the reactive oxygen metabolism of neutrophils than DCFH, i.e., by early oxygen metabolites like O2-, while DCFH is oxidized in part by H2O2 and phagosomal peroxidases. The differential oxidation of HE and DCFH during simultaneous cellular staining permits the analysis of up to three functionally different neutrophil populations in septic patients. This is of interest for the determination of disease-related alterations of oxygen metabolism in quiescent and stimulated leukocytes.