科研之友
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摘要
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 [C-14]-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.
- 出版日期1995-2
