Methods for distinguishing apoptotic from necrotic cells and measuring their clearance.

摘要: Three major morphological types of cell death can be distinguished: type I (apoptotic death), II (autophagic and III (necrotic death). Details the pathways apoptotic autophagic have been described, distinct biochemical markers identified. However, no surface or necrotic identified yet, only negative are available. These include absence parameters (caspase activation, cytochrome c release, oligonucleosomal DNA fragmentation) differential kinetics (phosphatidylserine exposure membrane permeabilization). Moreover, a confounding factor is that cells in phagocytosis proceed to secondary necrosis, which has many features primary cells. Secondary already gone through an stage, so it generally advisable research perform time parameters. This chapter concentrates on methods distinguish apoptosis from necrosis three different levels (morphological, biochemical, analysis cell-cell interactions) emphasizes combination several techniques correctly characterize type. First, we describe versus morphology by time-lapse microscopy, flow fluorocytometry, transmission electron microscopy. We also discuss various for permeability fluorocytometry), cellular such as fragmentation (flow caspase Bid cleavage, release (Western blotting). Next, how distinguished supernatant caspases, HMGB1, cytokeratin 18. Finally, interactions during quantitative method examining dead clearance fluorocytometry. A selection used study internalization mechanisms phagocytes engulf dying presented, scanning microscopy fluorescence