Methods for distinguishing apoptotic from necrotic cells and measuring their clearance.
Three major morphological types of cell death can be distinguished: type I (apoptotic cell death), type II (autophagic cell death), and type III (necrotic cell death). Details of the pathways of apoptotic and autophagic cell death have been described, and distinct biochemical markers have been identified. However, no distinct surface or biochemical markers of necrotic cell death have been identified yet, and only negative markers are available. These include absence of apoptotic parameters (caspase activation, cytochrome c release, and oligonucleosomal DNA fragmentation) and differential kinetics of cell death markers (phosphatidylserine exposure and cell membrane permeabilization). Moreover, a confounding factor is that apoptotic cells in the absence of phagocytosis proceed to secondary necrosis, which has many morphological features of primary necrotic cells. Secondary necrotic cells have already gone through an apoptotic stage, and so it is generally advisable in cell death research to perform time kinetics of cell death parameters. This chapter concentrates on methods that can distinguish apoptosis from necrosis on three different levels (morphological, biochemical, and analysis of cell-cell interactions) and emphasizes that only a combination of several techniques can correctly characterize cell death type. First, we describe analysis of apoptotic versus necrotic morphology by time-lapse microscopy, flow fluorocytometry, and transmission electron microscopy. We also discuss various biochemical techniques for analysis of cell surface markers (phosphatidylserine exposure versus cell permeability by flow fluorocytometry), cellular markers such as DNA fragmentation (flow fluorocytometry), caspase activation, Bid cleavage, and cytochrome c release (Western blotting). Next, we describe how primary and secondary necrotic cells can be distinguished by analysis of supernatant for caspases, HMGB1, and release of cytokeratin 18. Finally, we discuss cell-cell interactions during cell death and describe a quantitative method for examining dead cell clearance by flow fluorocytometry. A selection of techniques that can be used to study internalization mechanisms used by phagocytes to engulf dying cells is also presented, such as scanning and transmission electron microscopy and fluorescence microscopy.
Krysko
Dmitri V
DV
Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium.
Vanden Berghe
Tom
T
Parthoens
Eef
E
D'Herde
Katharina
K
Vandenabeele
Peter
P
D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D005434:Flow Cytometry; D006801:Humans; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D009336:Necrosis / Q000235:genetics / Q000503:physiopathology; D010718:Phosphatidylserines / Q000378:metabolism
D010718:Phosphatidylserines; D045304:Cytochromes c; D020169:Caspases