Morphological and biochemical characterization and analysis of apoptosis

Apoptosis is a form of programmed cell death serving physiologic and homeostatic functions. However, recent evidence implicating apoptosis in the etiology and pathophysiology of known human diseases, such as heart diseases, cancer, AIDS, and neurodegenerative and autoimmune diseases are continually surfacing. This has spawned the need for identifying which methods are the most effective and well accepted to decipher its presence in a variety of research settings. We have therefore detailed the morphology and biochemical features of apoptotic cell death, with an emphasis on discriminating it from necrosis. In addition, we describe specific and selective techniques which are optimal to target hallmark apoptotic features, such as microscopy, Annexin V labeling, in situ nick-end labeling (TUNEL), and DNA fragmentation analysis by gel electrophoresis and ELISA for oligonucleosome-sized DNA. The advantages and disadvantages of each technique are discussed, as well as their experimental importance relative to one another. The methods have been described in a stepwise fashion, and can readily be applied in the majority of cell systems. Whether working on the tissue or single cell level, these methods are highly effective in qualifying and quantifying apoptosis. The application of these methods in conjunction with molecular techniques can further delineate the underlying mechanisms of apoptosis.