Analyzing markers of apoptosis in vitro.

Cell death by apoptosis was first identified based on morphological changes reproduced with great fidelity in cells of widely different origin when exposed to a death stimulus. These changes include condensation of the cytosol and the nuclear chromatin, blebbing of the plasma membrane, and cell fragmentation into corpses that are engulfed by neighboring cells. Apoptotic cells demonstrate various levels of DNA fragmentation and exposed phosphatidylserine on the outer leaflet of their plasma membrane. Most apoptotic pathways converge on the mitochondria, inducing the disruption of the mitochondrial trans-membrane potential and the release of soluble molecules from mitochondrial inter-membrane space. One of these molecules is cytochrome c, which, in the cytosol, activates proteases of the caspase family. This chapter suggests methods to identify these characteristic morphological and biochemical events, and cell-free systems that can be used to identify the molecular pathways leading to the death phenotype.