Burow S, Valet G
Eur J Cell Biol. 1987 Feb;43(1):128-33.
A standardized four-step assay for the flow cytometric determination of the oxidative activity of human polymorphonuclear leukocytes (PMNL) from normal human individuals and from septic patients was developed, using 2,7-dichlorofluorescin-diacetate (DCFH-DA) as indicator for the intracellular formation of H2O2 and free radicals. Spontaneous H2O2 and free radical formation was measured by preincubation of buffy coat PMNLs from fresh peripheral venous blood at 37 degrees C and pH 7.4 with 10 microM DCFH-DA. Intracellular peroxidase activity was determined by addition of 1 mM external H2O2 to this assay. A maximum of granulocyte oxidative burst activity was elicited by the addition of 150 nM phorbol-myristate-acetate (PMA). A physiological burst was generated by incubating buffy coat PMNLs together with E. coli bacteria. The DNA of dead cells was in all instances simultaneously counterstained with propidium iodide (PI). Quiescent or H2O2 or bacteria treated granulocytes moved as a single cell cluster to higher fluorescences. Stimulation with PMA, in contrast, generated always a bimodal distribution of granulocyte fluorescence with the high activity cell cluster being approximately sevenfold more active than the low activity cell cluster. Roughly half of the granulocytes in normal individuals had high fluorescence. An increase of the high activity granulocytes was observed in septic patients. Model experiments with the nonfluorescent DCFH-DA cleavage product DCFH (2,7-dichlorofluorescin) showed that DCFH was quickly photo-oxidized to fluorescent DCF (2,7-dichlorofluorescein) by UV-light and to a lower degree by daylight. DCFH even slowly autooxidized in the dark.(ABSTRACT TRUNCATED AT 250 WORDS)
开发了一种标准化的四步检测法，用于通过流式细胞术测定正常人和脓毒症患者的人类多形核白细胞（PMNL）的氧化活性，使用2,7-二氯荧光素二乙酸酯（DCFH-DA）作为细胞内H2O2和自由基形成的指示剂。通过将新鲜外周静脉血的血沉棕黄层PMNL在37℃和pH 7.4下与10μM DCFH-DA预孵育来测量自发的H2O2和自由基形成。通过向该检测中加入1 mM外部H2O2来测定细胞内过氧化物酶活性。加入150 nM佛波酯-肉豆蔻酸酯-乙酸酯（PMA）可引发最大程度的粒细胞氧化爆发活性。通过将血沉棕黄层PMNL与大肠杆菌一起孵育产生生理性爆发。在所有情况下，死细胞的DNA都同时用碘化丙啶（PI）进行复染。静止的或经H2O2或细菌处理的粒细胞作为单个细胞簇向更高荧光移动。相比之下，用PMA刺激总是产生粒细胞荧光的双峰分布，高活性细胞簇的活性比低活性细胞簇高约七倍。正常个体中约一半的粒细胞具有高荧光。在脓毒症患者中观察到高活性粒细胞增加。用非荧光性DCFH-DA裂解产物DCFH（2,7-二氯荧光素）进行的模型实验表明，DCFH在紫外线下迅速光氧化为荧光性DCF（2,7-二氯荧光素），在日光下光氧化程度较低。DCFH甚至在黑暗中缓慢自动氧化。（摘要截短于250字）
