Bass D A, Parce J W, Dechatelet L R, Szejda P, Seeds M C, Thomas M
J Immunol. 1983 Apr;130(4):1910-7.
We have developed a quantitative assay to monitor the oxidative burst (H2O2 production) of polymorphonuclear leukocytes (PMNL) using single cell analysis by flow cytometry, and have examined whether PMNL respond to membrane stimulation with an all-or-none oxidative burst. During incubation with normal neutrophils, dichlorofluorescin diacetate diffused into the cells, was hydrolyzed to 2',7'-dichlorofluorescin (DCFH) and was thereby trapped within the cells. The intracellular DCFH, a nonfluorescent fluorescein analogue, was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by PMNL stimulated by phorbol myristate acetate (PMA). That the oxidative product was DCF was shown by excitation/emission spectra and by mass spectrometry of the product from PMA-stimulated PMNL. Normal resting and PMA-stimulated PMNL oxidized 6.9 +/- 0.7 and 160 +/- 13 attomoles DCF per cell, respectively, in 15 min. Absence of calcium and magnesium ions and/or addition of 2 mM EDTA did not inhibit DCF formation by PMNL stimulated by 100 ng/ml PMA. Since EDTA prevented aggregation of PMNL (even when stimulated by 100 ng/ml PMA), which would prevent accurate flow cytometric analysis, further experiments were performed with EDTA in the medium. A close correlation between average DCFH oxidation and hexose monophosphate shunt stimulation was demonstrated using cells from patients whose PMNL had oxidative metabolic defects of varying severity. Intracellular DCFH was also oxidized by reagent H2O2 or oxygen derivatives generated by glucose oxidase + glucose or by xanthine oxidase + acetaldehyde; DCFH oxidation by these systems was inhibited by catalase but unchanged by superoxide dismutase. The data indicate that the DCFH oxidation assay is quantitatively related to the oxidative metabolic burst of PMNL, and they strongly suggest that the reaction is mediated by H2O2 generated by the PMNL. Incubation of PMNL with varying concentrations of PMA caused graded responses by all PMNL present; i.e., 1 ng/ml PMA caused a mean response of 34% maximal with a single population of responding PMNL (rather than 66% resting and 34% fully stimulated as predicted by the all-or-none hypothesis). Thus, with these assay conditions, oxidative product formation by PMNL occurs as a graded response to membrane stimulation by PMA.
我们开发了一种定量检测方法，通过流式细胞术进行单细胞分析来监测多形核白细胞（PMNL）的氧化爆发（过氧化氢生成），并研究了PMNL是否以全或无的氧化爆发方式对膜刺激作出反应。在与正常中性粒细胞孵育期间，二氯荧光素二乙酸酯扩散进入细胞，被水解为2',7'-二氯荧光素（DCFH），并因此被困在细胞内。细胞内的DCFH是一种无荧光的荧光素类似物，被佛波酯肉豆蔻酸酯（PMA）刺激的PMNL氧化为高荧光的2',7'-二氯荧光素（DCF）。通过激发/发射光谱以及PMA刺激的PMNL产物的质谱分析表明氧化产物为DCF。正常静息和PMA刺激的PMNL在15分钟内分别氧化6.9±0.7和160±13阿托摩尔的DCF/细胞。缺乏钙和镁离子以及/或者添加2 mM乙二胺四乙酸（EDTA）并不抑制100 ng/ml PMA刺激的PMNL形成DCF。由于EDTA可防止PMNL聚集（即使在100 ng/ml PMA刺激下），而这会妨碍准确的流式细胞术分析，因此在培养基中加入EDTA进行了进一步实验。使用患有不同严重程度氧化代谢缺陷的PMNL的患者的细胞，证明了平均DCFH氧化与磷酸己糖旁路刺激之间存在密切相关性。细胞内的DCFH也会被试剂过氧化氢或葡萄糖氧化酶+葡萄糖或黄嘌呤氧化酶+乙醛产生的氧衍生物氧化；这些系统引起的DCFH氧化被过氧化氢酶抑制，但不受超氧化物歧化酶影响。数据表明DCFH氧化检测与PMNL的氧化代谢爆发在数量上相关，并且强烈表明该反应由PMNL产生的过氧化氢介导。用不同浓度的PMA孵育PMNL会使所有存在的PMNL产生分级反应；即，1 ng/ml PMA会使单一反应性PMNL群体产生平均为最大反应34%的反应（而不是如全或无假设所预测的66%静息和34%完全刺激）。因此，在这些检测条件下，PMNL的氧化产物形成是对PMA膜刺激的分级反应。
