MULTIPARAMETRIC FLOW CYTOMETRIC ANALYSIS OF RADIATION-INDUCED MICRONUCLEI IN MAMMALIAN-CELL CULTURES
MULTIPARAMETRIC FLOW CYTOMETRIC ANALYSIS OF RADIATION-INDUCED MICRONUCLEI IN MAMMALIAN-CELL CULTURES
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DOI：
10.1002/cyto.990130114
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发表时间：
1992-01-01
期刊：
影响因子：
--
通讯作者：
NUSSE, M
中科院分区：
文献类型：
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作者：
SCHREIBER, GA;BEISKER, W;NUSSE, M
提出了一种新的流式细胞术方法，可以高精度量化哺乳动物细胞培养物中辐射诱导的微核的频率。制备用溴化乙锭和 Hoechst 33258 染色的主核和微核悬浮液后，使用前向光散射和由 UV、488 nm 激发的三种荧光发射强度以及通过从 Hoechst 33258 到溴化乙锭的能量转移，在流式细胞仪中同时测量两种类型的颗粒。通过计算不同荧光的比率，将与微核分布重叠的非特异性碎片，特别是在低荧光强度区域中的非特异性碎片与微核区分开。用这种新技术测量的辐射诱导微核的频率与传统显微镜获得的结果非常吻合。发现通过该技术鉴定的微核DNA含量的下限约为G1期细胞核DNA含量的0.5%-0.75%。测量了两种不同小鼠细胞系的剂量效应曲线和微核的时间依赖性诱导。
A new flow cytometric method is presented that quantifies the frequency of radiation-induced micronuclei in mammalian cell cultures with high precision. After preparing a suspension of main nuclei and micronuclei stained with ethidium bromide and Hoechst 33258, both types of particles are measured simultaneously in a flow cytometer using forward light scatter and three fluorescence emission intensities excited by UV, 488 nm, and by energy transfer from Hoechst 33258 to ethidium bromide. Nonspecific debris overlapping the micronucleus distribution especially in the low fluorescence intensity region was discriminated from micronuclei by calculating ratios of the different fluorescences. The frequencies of radiation-induced micro-nuclei measured with this new technique agreed well with results obtained by conventional microscopy. The lower limit of the DNA content of micronuclei identified by this technique was found to be about 0.5%-0.75% of the DNA content of G1-phase nuclei. Dose effect curves and the time-dependent induction of micro-nuclei were measured for two different mouse cell lines.