Ruch W, Cooper P H, Baggiolini M
J Immunol Methods. 1983 Oct 28;63(3):347-57. doi: 10.1016/s0022-1759(83)80008-8.
A simple and sensitive method for measurement of the release of H2O2 from phagocytic cells is described. The assay is based on the H2O2-dependent oxidation of homovanillic acid (3-methoxy-4-hydroxyphenylacetic acid, HVA) to a highly fluorescent dimer (2,2'-dihydroxy-3,3'-dimethoxydiphenyl-5,5'-diacetic acid) which is mediated by horse-radish peroxidase. A linear relationship between fluorescence (lambda ex = 312 nm and lambda em = 420 nm) and amount of H2O2 was found in the range of 0.1-10 nmoles per 2.25 ml assay. The method was reliable for monitoring H2O2 production in large numbers of cell samples, as suspensions or monolayers, over periods of time extending between minutes and several hours. At concentrations optimal for detection of cellular release of H2O2, HVA and horse-radish peroxidase were devoid of cytotoxic effects. The time course of H2O2 release by mouse peritoneal and bone marrow-derived macrophages and by human neutrophils was determined following stimulation with zymosan particles or phorbol myristate acetate, and the dependence of H2O2 release on cell number and stimulus dosage was studied.
本文描述了一种用于测量吞噬细胞释放过氧化氢（H₂O₂）的简单且灵敏的方法。该检测方法基于在辣根过氧化物酶介导下，H₂O₂依赖的高香草酸（3 - 甲氧基 - 4 - 羟基苯乙酸，HVA）氧化为高荧光二聚体（2,2'-二羟基 - 3,3'-二甲氧基二苯基 - 5,5'-二乙酸）。在每2.25 ml检测体系中，0.1 - 10纳摩尔的H₂O₂范围内，荧光（激发波长λex = 312 nm，发射波长λem = 420 nm）与H₂O₂量之间呈线性关系。该方法可可靠地监测大量细胞样本（作为悬浮液或单层细胞）在数分钟至数小时内H₂O₂的产生情况。在检测细胞释放H₂O₂的最佳浓度下，HVA和辣根过氧化物酶无细胞毒性作用。在用酵母聚糖颗粒或佛波酯刺激后，测定了小鼠腹腔和骨髓来源的巨噬细胞以及人中性粒细胞释放H₂O₂的时间进程，并研究了H₂O₂释放对细胞数量和刺激剂量的依赖性。
