Cell death can be classified into a number of types including apoptotic, autophagic, necrotic and others. The hallmarks of apoptotic cell death include exposure of phosphatidyl serine on the extracellular face of the plasma membrane, activation of caspases, disruption of mitochondrial membrane potential, cell shrinkage, DNA fragmentation and DNA condensation. Necrotic cell death is characterized by cell swelling, as well as destruction of the plasma membrane and subcellular organelles, without nuclear fragmentation and condensation. Autophagic cell death is a degradative process involving sequestration of cellular components in double-membrane vesicles that fuse with lysosomes to form the autophagolysosomes. Autophagy is a survival strategy in situations of stress, but can also lead to non-apoptotic cell death. Due to the overall complexity of the cited death pathways, a sensitive assay capable of distinguishing between autophagy, apoptosis and necrosis is fundamental to further understanding of these pathophysiological processes. Thus, a three-parameter cell-based assay was developed utilizing a green-emitting fluorophore to highlight the various vacuolar components of the autophagy pathway, a violet-excitable fluorophore conjugate of annexin V to measure phosphatidyl serine exposure in early stage apoptosis, and a cell-impermeable red fluorescent DNA intercalation dye to measure membrane disintegration in late-stage apoptosis/necrosis. The assay was validated using a range of conditions known to modulate autophagy, apoptosis and necrosis pathways. The assay provides a rapid, information-rich read-out of the various cell death pathways by fluorescence microscopy and flow cytometry and should facilitate better drug activity profiling and clearer kinetic analysis of these fundamental processes in living cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2876. doi:10.1158/1538-7445.AM2011-2876

©2011 American Association for Cancer Research
2011