Abstract
A simple, sensitive, and reliable DNA diffusion assay for quantification of apoptosis is based on the principle that nuclear DNA of apoptotic cells have abundant alkali-labile sites and under alkaline conditions small pieces of DNA thus generated diffuse in agarose, giving the appearance of a halo if stained with a sensitive fluorescent dye such as YOYO-1. The protocol for detection of apoptosis described here is tested for estimation of apoptosis in human leukocytes (mostly neutrophils) incubated at 37°C for 24 h. Cells were mixed with agarose, microgels were made, and cells were lysed in a solution of high salt, detergents, and alkali. DNA was precipitated in microgels by ethanol and spermine. Staining of DNA was done with an intense fluorescent dye, YOYO-1. Apoptotic cells show a circular gradient of granular DNA with a dense central zone and a lighter and hazy outer zone, giving the overall appearance of a halo.
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Singh, N.P. (2005). Apoptosis Assessment by the DNA Diffusion Assay. In: Blumenthal, R.D. (eds) Chemosensitivity: Volume II. Methods in Molecular Medicine™, vol 111. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-889-7:055
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DOI: https://doi.org/10.1385/1-59259-889-7:055
Publisher Name: Humana Press, Totowa, NJ
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