Detection of tumor necrosis factor induced nuclear damage with p-phenylenediamine

Abstract

A simple technique has been used to observe the effects of rTNFα on nuclear morphology. Using the intercalating dye p-phenylenediamine to stain nuclei, we detected TNFα-induced nuclear alterations which characteristically occur during apoptosis in the TNFα sensitive U937 cell line. Nuclear alterations were visible prior to the loss of plasma membrane integrity and subsequent cell death. A subclone of U937 cells was isolated in which TNFα failed to alter either cell viability or nuclear morphology. TNFα resistant U937 cells, however, retained the ability to bind, internalize and degrade TNFα. These results suggest that nuclear damage induced by TNFα in sensitive U937 cells occurs early and precedes cell death as measured by dye exclusion assays. Staining cells with p-phynylenediamine and visualization with a 520 nm fluorescence filter provides a rapid and simple method to monitor apoptotic cell death.