Identification of apoptotic cells by formamide-induced DNA denaturation incondensed chromatin

In this article we describe a novel effect of formamide on DNA of apoptotic nuclei and present a method for specific detection of apoptotic cells base d on this effect. Our observations show that formamide induces DNA denatura tion in apoptotic nuclei but has no such effect on DNA of non-apoptotic cel ls. Formamide-induced DNA denaturation combined with detection of denatured DNA with a monoclonal antibody (MAb) against single-stranded DNA made it p ossible to specifically identify the apoptotic cells. This procedure produc ed intense staining of the condensed chromatin in the apoptotic nuclei. In contrast, necrotic cells from cultures treated with sodium azide, saponin, or hyperthermia did not bind this antibody, demonstrating the specificity o f the formamide-MAb assay for the apoptotic cells. However, TUNEL stained 9 0-100% of necrotic cells in all three models of necrosis. Because the MAb d id not stain cells with single- or double-stranded DNA breaks in the absenc e of apoptosis, we conclude that staining of the apoptotic nuclei is not in fluenced by DNA breaks and is induced by specific changes in condensed chro matin, such as damage to the DNA-histone interactions. Importantly, the for mamide-MAb technique identified apoptotic cells in frozen sections and in h istological sections of formalin-fixed, paraffin-embedded tissues.