Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle
Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle
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DOI：
10.1038/nprot.2013.011
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发表时间：
2013-03-01
期刊：
影响因子：
14.8
通讯作者：
Hengstschlaeger, Markus
中科院分区：
文献类型：
--
作者：
Rosner, Margit;Schipany, Katharina;Hengstschlaeger, Markus
该方案描述了一种方法，用于在正常细胞周期进程中使用未操作的异步细胞进行核质蛋白跟踪。与流行的传统方法相比，我们的方法不需要耗时，干扰细胞同步或分离。为此，我们选择了单细胞方法，并开发了适用于全细胞和分离的细胞核的流式细胞术测定。我们的方案涉及从全细胞中纯化细胞核的逐步生化分离程序，用于DNA和蛋白质双重标记细胞和细胞核的常规DNA和间接免疫染色技术，以及流式细胞术数据处理和计算的精细概念：通过DNA和细胞大小分析的特定组合，G1，进一步解剖细胞周期的S期和G2/M期，以建立细胞周期进程的高分辨率地图，细胞或细胞核中的蛋白质表达与此相关。在最后的数据分析步骤中，基于获得的全细胞和分离的细胞核的结果计算细胞周期相关的细胞质蛋白表达。至少需要8小时才能完成手术。由于该方法不需要限制细胞类型的预处理，因此可以容易地研究不同来源的许多细胞类型。人羊水干细胞、原代人成纤维细胞、永生化小鼠成纤维细胞和转化肿瘤细胞的分析效率相当，证明细胞间测定变异性低。
This protocol describes a method for nucleocytoplasmic protein tracking during normal cell cycle progression using unmanipulated, asynchronous cells. In contrast with prevalent traditional methods, our approach does not require time-consuming, perturbing cell synchronization or separation. To this end, we chose a single-cell approach and developed a flow cytometry assay that is applied to whole cells and isolated nuclei. Our protocol involves a stepwise biochemical fractionation procedure to purify nuclei from whole cells, conventional DNA and indirect immunostaining techniques for the dual labeling of cells and nuclei for DNA and protein, and a refined concept of flow cytometric data processing and calculation: through the specific combination of DNA and cell size analyses, G1, S and G2/M phases of the cell cycle are further dissected to establish a high-resolution map of cell cycle progression, to which protein expression in cells or nuclei is correlated. In a final data analysis step, cell cycle-related, cytoplasmic protein expression is calculated on the basis of results obtained for whole cells and isolated nuclei. A minimum of 8 h is required to complete the procedure. As the approach does not require cell type-restricting pretreatments, numerous cell types of different origin can be readily studied. Human amniotic fluid stem cells, primary human fibroblasts, immortalized mouse fibroblasts and transformed tumor cells are analyzed at comparable efficiencies, demonstrating low intercell assay variability.