Huang C J, Severin E
Institut für Strahlenbiologie der Universität Münster, Germany.
Acta Histochem. 1993 Feb;94(1):33-45. doi: 10.1016/S0065-1281(11)80337-5.
Flow cytometric measurements of the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, glycerol-3-phosphate dehydrogenase, succinate dehydrogenase and glutamate dehydrogenase in single Ehrlich ascites tumour cells are described using a tetrazolium salt/fluorescent formazan reaction. Applying cyano-ditolyl-tetrazolium chloride (CTC) as redox dye indicating enzyme reaction, and DAPI as a fluorochrome for nuclear DNA staining, the bivariate flow cytometric assay of enzyme activity and cell cycle analysis was established. Furthermore, adopting the calibration procedure reported formerly, consisting of biochemical determination and flow cytometry of the same sample performed parallelly, the enzyme activities were expressed in biochemical units. The dehydrogenase activities found in Ehrlich ascites cells were 97.5 fmol H2 per average positive cell during 5 min for lactate dehydrogenase, 69.0, 10.6, 25.3, 29.7, and 19.0 fmol H2 per average positive cell during 20 min for glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, glycerol-3-phosphate dehydrogenase, succinate dehydrogenase and glutamate dehydrogenase, respectively. This quantitative procedure can offer an alternative analytic tool for enzyme cytology.
本文描述了使用四唑盐/荧光甲臜反应对单个艾氏腹水肿瘤细胞中乳酸脱氢酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、3-磷酸甘油脱氢酶、琥珀酸脱氢酶和谷氨酸脱氢酶的活性进行流式细胞术测量。应用氯化氰二苯并噻唑鎓（CTC）作为指示酶反应的氧化还原染料，并用4',6-二脒基-2-苯基吲哚（DAPI）作为核DNA染色的荧光染料，建立了酶活性的双变量流式细胞术测定和细胞周期分析方法。此外，采用先前报道的校准程序，即对同一样品同时进行生化测定和流式细胞术分析，酶活性以生化单位表示。艾氏腹水细胞中发现的脱氢酶活性分别为：乳酸脱氢酶在5分钟内每个平均阳性细胞97.5 fmol H2；葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、3-磷酸甘油脱氢酶、琥珀酸脱氢酶和谷氨酸脱氢酶在20分钟内每个平均阳性细胞分别为69.0、10.6、25.3、29.7和19.0 fmol H2。这种定量方法可为酶细胞学提供一种替代分析工具。
