title: Cell cycle staging of individual cells by fluorescence microscopy.
abstract: 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.
authors: National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors: Vassilis
authors: V
authors: Roukos
authors: 1] National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA. [2] High-Throughput Imaging Facility, National Cancer Institute, NIH, Bethesda, Maryland, USA.
authors: Gianluca
authors: G
authors: Pegoraro
authors: 1] National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA. [2] PerkinElmer Health Sciences Inc., Waltham, Maryland, USA.
authors: Ty C
authors: TC
authors: Voss
authors: National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors: Tom
authors: T
authors: Misteli
mesh_terms: D000465:Algorithms; D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D005456:Fluorescent Dyes; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007211:Indoles / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods; D012984:Software
chemical_list: D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI
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