Evaluation of eight fluorochrome combinations for simultaneous DNA-protein flow analyses.
Evaluation of eight fluorochrome combinations for simultaneous DNA-protein flow analyses.
复制标题
评估用于同步 DNA-蛋白质流分析的八种荧光染料组合。
作者：
M. Stöhr;M. Vogt;Manfred Knobloch;R. L. Vogel;G. Futterman
用光谱法和流动显微荧光法评价了8种用于DNA-蛋白质同时染色的荧光染料组合：碘化丙啶（PI）与异硫氰酸荧光素（FITC）、荧光胺（FC）和丹磺酰氯（DANS），二脒基苯基吲哚（DAPII）与磺罗丹明（SR 101）、四甲基罗丹明异硫氰酸酯（TRITC）和硝基苯并二唑（NBD）;吖啶黄（AF）与二苯乙烯异硫氰酸酯磺酸（SITS）和DAPI。在研究中使用了三种不同的实验肿瘤细胞系。同时进行了DNA-蛋白质分析与新开发的HEIFAS仪器。在光谱上，两组染料分别根据它们在400 nm以下和450 nm以上的激发最大值来区分。DANS和NBD被认为是不令人满意的，就其蛋白质分布获得的流动分析。染料组合中涉及的其余染色剂显示细胞DNA和蛋白质含量的可比较的流动分布。关于染色方案中涉及的制备时间和离心步骤的数量，以及所用染料的稳定性，DAPI-SR 101方法被证明是最快和最简单的。通过这种组合，可以在30分钟内同时进行DNA和蛋白质流分析。
Eight fluorescent dye combinations for simultaneous DNA-protein staining have been evaluated spectroscopically and flow microfluorometrically: propidium iodide (PI) with fluoresceinisothiocyanate (FITC), fluorescamine (FC), and dansylchloride (DANS); diamidinophenylindole (DAPII) with sulphorhodamin (SR101), tetramethylrhodamin isothiocyanate (TRITC), and nitrobenzodiazole (NBD); acriflavine (AF) with stilbene isothiocyanate sulphonic acid (SITS), and DAPI. Three different experimental tumor cell lines have been employed in the investigations. Simultaneous DNA-protein analyses have been carried out with the newly developed HEIFAS instrument. Spectroscopically two groups of dyes were distinguishable according to their excitation maximum below 400 nm and above 450 nm respectively. DANS and NBD were found to be unsatisfactory with respect to their protein distributions obtained by flow analysis. The remaining stains involved in the dye combination revealed comparable flow distributions of the cellular DNA and protein content. With respect to preparation time and number of centrifugal steps involved in the staining protocols, and in connection with the stability of the dye used, the DAPI-SR101 method proved to be fastest and easiest. With this combination DNA and protein flow analysis can be performed simultaneously within 30 min.