Detection of apoptosis-associated DNA fragmentation using a rapid and quantitative filter elution assay

DNA degradation to oligonucleosome size fragments is one of the cellular markers of apoptosis. Based on DNA filter elution methodology, we have designed a simple assay to monitor apoptosis-associated DNA fragmentation from cultured cells or from a reconstituted cell-free system. The cells are prelabeled with [¹⁴C]-thymidine for one cell cycle and chased in nonradioactive medium for a few hours in order to allow incorporation of the radiolabeled thymidine in high molecular weight DNA. After the apoptosis-inducing treatment, cells (or subcellular fractions of the cell-free system) are deposited onto a protein-adsorbant filter and washed with physiological saline. Lysis is then performed with a mild detergent (sarkosyl) and 2 M salt. The lysis fraction is collected, counted, and computed to calculate the fraction of DNA in the lysis fraction. In normal cells more than 90% of the DNA remains on the filter, while in apoptotic cells the kinetics of DNA fragmentation can be monitored and more than 80% of the counts can be found in the lysis. The DNA filter elution assay is sensitive, quantitative, and rapid. By using different types of filters and lysis solution (± proteinase), the protein-bonding to the DNA fragments can be determined. Some applications of this assay to study the effects and mechanisms of action of new therapeutic drugs are presented and discussed. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.