Apoptosis Assessment by the DNA Diffusion Assay

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.