Henderson L M, Chappell J B
Department of Biochemistry, School of Medical Sciences, University of Bristol, England.
Eur J Biochem. 1993 Nov 1;217(3):973-80. doi: 10.1111/j.1432-1033.1993.tb18328.x.
Imaging techniques, such as confocal microscopy and fluorescent activated cells scan are facilitating the study of responses at the single-cell level. Superoxide is reported to oxidise the non-fluorescent dihydrorhodamine 123 (DHR) to rhodamine 123. The generation of rhodamine 123 by human neutrophils, stimulated by the phorbol ester phorbol 12-myristate 13-acetate was inhibited slowly by diphenylene iodonium and rapidly by azide, but not by superoxide dismutase. In the absence of enzymes H2O2 (but not O2-.) oxidised DHR slowly but the rate was greatly enhanced by peroxidases. The rhodamine 123 generated by phorbol-ester-stimulated neutrophils was observed to be located within the cell despite the fact that neutrophils failed to accumulate external rhodamine 123. This stimulated rise in cellular fluorescence was eliminated by excess extracellular catalase. It appears that H2O2, released on the outside, crosses the plasma membrane where oxidation of DHR is catalysed by cellular peroxidases. Since in a mixed population DHR failed to distinguish between O2-.-producing and non-producing HL60 cells it is not a suitable probe for single-cell observations. We conclude that DHR oxidation reports only the presence of H2O2 and intracellular peroxidases, and not the generation of O2-. by any one cell. Only peroxidase-containing cells fluoresce.
成像技术，如共聚焦显微镜和荧光激活细胞扫描，正在促进单细胞水平反应的研究。据报道，超氧化物可将非荧光性的二氢罗丹明123（DHR）氧化为罗丹明123。佛波酯佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯刺激人中性粒细胞产生罗丹明123的过程，被二亚苯基碘鎓缓慢抑制，被叠氮化物迅速抑制，但不被超氧化物歧化酶抑制。在没有酶的情况下，H2O2（而非O2-）缓慢氧化DHR，但过氧化物酶可大大提高其氧化速率。尽管中性粒细胞不能积累细胞外的罗丹明123，但观察到佛波酯刺激的中性粒细胞产生的罗丹明123位于细胞内。细胞外过量的过氧化氢酶消除了这种刺激引起的细胞荧光增强。似乎释放到细胞外的H2O2穿过质膜，在质膜上DHR的氧化由细胞内过氧化物酶催化。由于在混合群体中，DHR无法区分产生O2-的和不产生O2-的HL60细胞，因此它不是用于单细胞观察的合适探针。我们得出结论，DHR氧化仅报告H2O2和细胞内过氧化物酶的存在，而不报告任何一个细胞产生O2-的情况。只有含过氧化物酶的细胞会发出荧光。
