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Curr Protoc Immunol. 2017 Nov 1;119:5.7.1-5.7.20. doi: 10.1002/cpim.36.

Analysis of Cellular DNA Content by Flow Cytometry.

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Department of Pathology and Brander Cancer Research Institute, New York Medical College, Valhalla, New York.
Hematology/Oncology, Case Western Reserve University Cleveland, Ohio.


Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content.


4′6-diamidino-2-phenylindole; DAPI; DNA index; DNA ploidy; Hoechst 33342; PI; cell cycle; propidium iodide

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