Roukos Vassilis, Pegoraro Gianluca, Voss Ty C, Misteli Tom
National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA.
1] National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA. [2] High-Throughput Imaging Facility, National Cancer Institute, NIH, Bethesda, Maryland, USA.
Nat Protoc. 2015 Feb;10(2):334-48. doi: 10.1038/nprot.2015.016. Epub 2015 Jan 29.
Progression through the cell cycle is one of the most fundamental features of cells. Studies of the cell cycle have traditionally relied on the analysis of populations, and they often require specific markers or the use of genetically modified systems, making it difficult to determine the cell cycle stage of individual, unperturbed cells. We describe a protocol, suitable for use in high-resolution imaging approaches, for determining cell cycle staging of individual cells by measuring their DNA content by fluorescence microscopy. The approach is based on the accurate quantification by image analysis of the integrated nuclear intensity of cells stained with a DNA dye, and it can be used in combination with several histochemical methods. We describe and provide the algorithms for two automated image analysis pipelines and the derivation of cell cycle profiles with both commercial and open-source software. This 1-2-d protocol is applicable to adherent cells, and it is adaptable for use with several DNA dyes.
细胞周期进程是细胞最基本的特征之一。传统上，细胞周期研究依赖于群体分析，通常需要特定标记或使用基因改造系统，这使得确定单个未受干扰细胞的细胞周期阶段变得困难。我们描述了一种适用于高分辨率成像方法的方案，通过荧光显微镜测量单个细胞的DNA含量来确定其细胞周期阶段。该方法基于对用DNA染料染色的细胞的细胞核积分强度进行图像分析的精确量化，并且可以与多种组织化学方法结合使用。我们描述并提供了两种自动图像分析流程的算法，以及使用商业和开源软件推导细胞周期图谱的方法。这个1-2天的方案适用于贴壁细胞，并且适用于多种DNA染料。
