Distinguishing between apoptosis, necrosis, necroptosis and other cell death modalities.

This issue of Methods contains a series of articles that cover the major techniques used to measure apoptosis, necrosis and variations thereof, at the population, single cell, organelle and molecular level. Although we used to think of apoptosis as programmed or regulated cell death and necrosis as strictly non-programmed or unregulated cell death [1,2], data have emerged in recent years which suggest that the boundaries between these two modes of cell death can become blurred in certain situations [3,4]. To summarize, for those that are new to the field, apoptosis is a mode of cell death that is characterized by a series of morphological and biochemical alterations to the cell architecture that package a cell up for removal by cells with phagocytic capacity [5]. Crucially, apoptotic cells are recognized by phagocytes and are engulfed before they leak their contents. Thus, apoptosis ensures that when a cell needs to be removed from a tissue, this occurs in an orderly fashion that minimizes disruption to neighboring cells. The major consideration during apoptosis is that intracellular contents do not leak into the extracellular space because this could (a) damage surrounding cells, and (b) trigger inflammation through release of molecules with immune-activating activity, the so-called ‘alarmins’ [5]. Thus, apoptosis is largely concerned with avoiding disturbance to tissues in which there is ongoing homeostatic cell death. Most of the biochemical and morphological changes that typify apoptosis are the consequence of activation of a subset of the caspase family of proteases (caspases 3, 6, 7, 8 and 9) [1,5]. The latter caspases operate similar to a controlled demolition squad, coordinating the packaging and disposal of cells in a manner that minimizes damage to neighbors and the initiation of inflammation [5]. Methods for measuring apoptosis typically rely on the detection of caspase-dependent events, such as exposure of plasma membrane phosphatidylserine [6], that precede uptake of vital dyes such as trypan blue or propidium iodide. Alternatively, the striking morphological features of apoptotic cells (such as compaction and fragmentation of the cell nucleus), which are also effected through caspase activation, are still highly relevant for methods for detecting this mode of cell death. In contrast to apoptosis, necrosis is generally uncontrolled and involves the sudden loss of membrane integrity, release of extracellular contents, leading to activation of the immune system and extensive inflammation [2,4]. Necrosis is typically not associated with caspase activation, although the exception to this is where cell death follows aggressive activation of the inflammatory subset of caspases (caspases 1, 4 and 5), a mode of cell death termed