Isolation of chromatin fragments released by apoptotic cells

Abstract A two-step procedure for isolation of chromatin fragments released from apoptotic cells is described. The first step includes extraction of the chromatin fragments by a hypotonic treatment of isolated nuclei. The second step provides stabilization of the extracted chromatin fragments and their collection by a high-speed centrifugation. The procedure ensures not only a complete extraction and collection of the chromatin fragments but also intactness of their components. A number of control experiments confirmed this statement: they showed that the percentage of recovered fragmented DNA does not exceed 10% from the total DNA of apoptotic cells and that it is specific in both nature and chromosomal localization; the experiments reveal, moreover, that the protein components of the fragments including the well known histone and non-histone species possess also ability to support in in vitro conditions their specific phosphorylation. Developed for recovery of chromatin fragments from mouse erythroleukemia cells, spontaneously entering apoptosis, the procedure is practically applicable for all eukaryotic cell systems sharing programmable death.