A simple method for accurate estimation of apoptotic cells

A simple, sensitive, and reliable "DNA diffusion" assay for the quantificat ion of apoptosis is described. Human lymphocytes and human lymphoblastoid c ells, MOLT-4, were exposed to 0, 12,5, 25, 50, or 100 rad of X-rays. After 24 h of incubation, cells mere mixed with agarose, microgels were made, and cells mere lysed in high salt and detergents. DNA was precipitated in micr ogels by ethanol. Staining of DNA was done with an intense fluorescent dye, YOYO-1. Apoptotic cells show a halo of granular DNA with a hazy outer boun dary. Necrotic cells, resulting from hyperthermia treatment, on the other h and, show an unusually large homogeneous nucleus with a clearly defined bou ndary. The number of cells with apoptotic and necrotic appearance can be sc ored and quantified by using a fluorescent microscope. Results were compare d with other methods of apoptosis measurement: morphological estimations of apoptosis and DNA ladder pattern formation in regular agarose gel electrop horesis. Validation of the technique was done using some known inducers of apoptosis and necrosis (hyperthermia, hydrogen peroxide, mitoxantrone, novo biocin, and sodium ascorbate). (C) 2000 Academic Press.