Methods to analyze cellular necroptosis

Abstract

Necroptosis is a mechanism of necrotic cell death induced by external stimuli in the form of death domain receptor (DR) engagement by their respective ligands, TNF-alpha, Fas ligand (FasL) and TRAIL, under conditions when apoptotic cell death execution is prevented, e.g. by caspase inhibitors. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. RIP1 kinase activity is a key step in the necroptosis pathway. We have previously identified specific and potent small-molecule inhibitors of necroptosis, necrostatins, which efficiently prevent execution of this form of cell death. Herein, we describe the methods to analyze cellular necroptosis, and the methods to analyze the inhibitory effects of anti-necroptosis compounds (necrostatin-1).