Induction and Detection of Necroptotic Cell Death in Mammalian Cell Culture.

The study of necroptosis is a rapidly growing field in current research of cell death mechanisms and cancer treatment strategies. While apoptotic cells can be reliably identified via annexin V assay, necroptosis is not associated with exposure of easily detectable markers. The most reliable way to identify necroptotic events is immunochemical detection of active phosphorylated RIPK1, RIPK3, and MLKL proteins facilitating necroptosis execution. This chapter describes a detailed protocol on necroptosis induction in human colon adenocarcinoma HT-29 cells, preparation of various positive and negative controls, detection of necroptosis mediator proteins via Western Blot analysis, and interpretation of results. This protocol allows reliable and specific detection of necroptosis in cell culture or tissue samples, and it provides a well-established model suitable for detailed studies of necroptosis molecular mechanisms in vitro.