Tools in the art of studying necroptosis

Necroptosis is a more recently described form of regulated cell death (RCD) that occurs in a caspase-independent manner. This is a lytic form of cell death in which the cellular contents are released and these contents serve as damage-associated molecular patterns (DAMPs). DAMPs are endogenous ligands for pattern recognition receptors and therefore necroptosis is considered to be highly inflammatory and immunogenic. Members of the TNF family are the most well-studied triggers of necroptosis, though other immune receptors are also known to directly trigger this death pathway. Necroptosis is now defined to be dependent on the core signaling molecules of RIPK3 and MLKL. In the case of TNF, it also involves RIPK1, and induction of necroptosis often also requires inhibition of caspases. In this volume, a wide array of tools to study necroptosis are described. They include pharmacological, biochemical, cellular, and in vivo approaches. Combining multiple approaches in one’s study is ideal for generating conclusive evidence for the involvement of necroptosis. The protocols presented in this chapter are highly useful in studying necroptosis, whose physiological and pathophysiological roles remain incompletely understood.