Poletaev A I, Stepanova N G, Nikitin S M
Mol Biol (Mosk). 1988 Jul-Aug;22(4):1062-71.
The possibility of use of 7-amino-actinomycin D (7aAMD)--fluorescent analog of actinomycin D--as a specific dye for DNA staining in the suspended cells was studied by means of laser flow-cytometry. The optimal conditions for staining were obtained: 7aAMD concentration 10(-5) M, pH 7, staining time 20 min, 37 degrees C, ionic strength 0.15 M Na+. In this case the fluorescent signal is proportional to the DNA amount and coefficient of variation is about 0.03. The influence of the stepwise extraction of the proteins from chromatin also was studied. In the course of the salt deproteinization the fluorescence intensity gradually rose thus showing the increase of the binding sides-number. The deproteinization of cells nuclei by 0.1 HCl increased the number of binding sites 2.5 times more. It was shown that the incubation of cells with RNAse at elevated ionic strength (0.3-0.7 M NaCl) leads to an additional increase of the cell fluorescence and produces no effect at low and normal ionic strength. The deproteinizing effect of RNAse and its possible mechanism is discussed.
利用激光流式细胞术研究了放线菌素D的荧光类似物7-氨基放线菌素D（7aAMD）作为悬浮细胞中DNA特异性染色剂的可能性。获得了染色的最佳条件：7aAMD浓度为10^(-5) M，pH值为7，染色时间为20分钟，温度为37℃，离子强度为0.15 M Na+。在这种情况下，荧光信号与DNA量成正比，变异系数约为0.03。还研究了从染色质中逐步提取蛋白质的影响。在盐脱蛋白过程中，荧光强度逐渐升高，表明结合位点数量增加。用0.1 HCl对细胞核进行脱蛋白处理使结合位点数量增加了2.5倍。结果表明，在高离子强度（0.3 - 0.7 M NaCl）下用RNA酶孵育细胞会导致细胞荧光进一步增加，而在低离子强度和正常离子强度下则无影响。讨论了RNA酶的脱蛋白作用及其可能的机制。
