Preparation and bivariate analysis of suspensions of human chromosomes.
Preparation and bivariate analysis of suspensions of human chromosomes.
复制标题
人类染色体悬浮液的制备和双变量分析。
DOI：
10.1002/cyto.990060203
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Yu,LC
中科院分区：
文献类型：
--
作者：
vandenEngh,GJ;Trask,BJ;Gray,JW;Langlois,RG;Yu,LC
使用含有 KCl、MgSO4、二硫赤藓糖醇和 RNase 的 HEPES 缓冲低渗溶液（pH 8.0）从多种人类细胞类型中分离染色体。通过该程序分离的染色体可以用多种荧光染料染色，包括碘化丙啶、铬霉素 A3 和 Hoechst 33258。发现向染色染色体中添加柠檬酸钠可以提高总荧光分辨率。从人成纤维细胞株、人结肠癌细胞系和人外周血淋巴细胞培养物中分离的染色体获得了高质量的双变量 Hoechst 与铬霉素荧光分布。还从原代羊膜细胞培养物中获得了良好的流动核型。在不同时间和在四倍范围内变化的染料浓度下制作的给定细胞系的赫斯特与色霉素流核型，在染色体的相对峰位置上显示出很小的变化。使用此程序分离的染色体中 DNA 的大小范围为 20 至超过 50 KB。所述分离程序简单，可产生高质量的流式核型，可用于从临床样本中制备染色体。
Chromosomes were isolated from a variety of human cell types using a HEPES‐buffered hypotonic solution (pH 8.0) containing KCl, MgSO4, dithioerythritol, and RNase. The chromosomes isolated by this procedure could be stained with a variety of fluorescent stains including propidium iodide, chromomycin A3, and Hoechst 33258. Addition of sodium citrate to the stained chromosomes was found to improve the total fluorescence resolution. High‐quality bivariate Hoechst vs. chromomycin fluorescence distributions were obtained for chromosomes isolated from a human fibroblast cell strain, a human colon carcinoma cell line, and human peripheral blood lymphocyte cultures. Good flow karyotypes were also obtained from primary amniotic cell cultures. The Hoechst vs. chromomycin flow karyotypes of a given cell line, made at different times and at dye concentrations varying over fourfold ranges, show little variation in the relative peak positions of the chromosomes. The size of the DNA in chromosomes isolated using this procedure ranges from 20 to over 50 kilobases. The described isolation procedure is simple, it yields high‐quality flow karyotypes, and it can be used to prepare chromosomes from clinical samples.