Butler W B
Anal Biochem. 1984 Aug 15;141(1):70-3. doi: 10.1016/0003-2697(84)90426-3.
A procedure is described for preparing nuclei from cells in monolayer culture so that they may be counted using an electronic particle counter. It takes only 10 to 15 min, and consists of swelling the cells in hypotonic buffer and then lysing them with the quaternary ammonium salt, ethylhexadecyldimethylammonium bromide. The cells are completely lysed, yielding a suspension of clean single nuclei which is stable, free of debris, and easily counted. The method was developed for a cell line of epithelial origin (MCF-7), which is often difficult to trypsinize to single cells. It works equally well at all cell densities up to and beyond confluence, and has been used with a variety of cells in culture, including 3T3 cells, bovine macrophages, rat mammary epithelial cells, mouse mammary tumor cell lines, and human fibroblasts. The size of the nuclei produced by this procedure is related to their DNA content, and the method is thus suitable for following cultures of synchronized cells through the cell cycle, and for performing differential counts of cells with substantial differences in DNA content.
本文描述了一种从单层培养细胞中制备细胞核的方法，以便使用电子粒子计数器对其进行计数。该方法仅需10至15分钟，包括将细胞在低渗缓冲液中膨胀，然后用季铵盐十六烷基二甲基乙基溴化铵裂解细胞。细胞被完全裂解，产生纯净的单核悬浮液，该悬浮液稳定、无碎片且易于计数。该方法是针对上皮来源的细胞系（MCF-7）开发的，该细胞系通常难以用胰蛋白酶消化成单细胞。在直至汇合及汇合后所有细胞密度下该方法均同样有效，并且已用于多种培养细胞，包括3T3细胞、牛巨噬细胞、大鼠乳腺上皮细胞、小鼠乳腺肿瘤细胞系和人成纤维细胞。通过该方法产生的细胞核大小与其DNA含量相关，因此该方法适用于跟踪同步化细胞在细胞周期中的培养情况，以及对DNA含量有显著差异的细胞进行鉴别计数。
