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Nat Protoc. 2015 Feb;10(2):334-48. doi: 10.1038/nprot.2015.016. Epub 2015 Jan 29.

Cell cycle staging of individual cells by fluorescence microscopy.

Author information

1
National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA.
2
1] National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA. [2] High-Throughput Imaging Facility, National Cancer Institute, NIH, Bethesda, Maryland, USA.
3
1] National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA. [2] PerkinElmer Health Sciences Inc., Waltham, Maryland, USA.

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.

PMID:
25633629
PMCID:
PMC6318798
DOI:
10.1038/nprot.2015.016
[Indexed for MEDLINE]
Free PMC Article

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