Ulrich W
Institute of Genetics, University of Hohenheim, Stuttgart, Germany.
Biotech Histochem. 1992 Mar;67(2):73-8. doi: 10.3109/10520299209110011.
A 2-step staining procedure is presented for simultaneous measurement of nuclear DNA and protein content in higher plants by flow cytometry. To release nuclei, plant tissues were chopped and stirred in the presence of the DNA specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) and the nonionic detergent Triton X-100. Plant protoplasts were stirred in the DAPI dye solution with the detergent. After a short incubation period a second dye solution containing DAPI and the protein fluorochrome sulforhodamine 101 (SR 101) without detergent was added. Following another incubation, and after filtration through nylon gauze, the highly fluorescent nuclei were analyzed with an impulse cytophotometer. Accurate bivariate DNA-protein histograms were obtained with CV-values of about 2% or less for the 2C-peak of the univariate DNA parameter. The method presented here can be used for basic and applied cytogenetic studies of higher plants, for characterization of subcompartments of the cell cycle phases, or for examination of heterogeneity in plant tissues.
本文介绍了一种两步染色程序，用于通过流式细胞术同时测量高等植物细胞核DNA和蛋白质含量。为了释放细胞核，将植物组织在DNA特异性荧光染料4',6-二脒基-2-苯基吲哚（DAPI）和非离子去污剂Triton X-100存在的情况下切碎并搅拌。将植物原生质体在含有去污剂的DAPI染料溶液中搅拌。短时间孵育后，加入第二种不含去污剂的含有DAPI和蛋白质荧光染料磺基罗丹明101（SR 101）的染料溶液。再次孵育后，通过尼龙纱布过滤，用脉冲细胞光度计分析高荧光细胞核。对于单变量DNA参数的2C峰，获得了准确的双变量DNA-蛋白质直方图，CV值约为2%或更低。本文介绍的方法可用于高等植物的基础和应用细胞遗传学研究、细胞周期相亚区室的表征或植物组织异质性的检测。
