Protocol for in vitro profiling of key TNF downstream signaling pathways involved in cell death and pro-survival signaling in cancer cells.

Tumor necrosis factor (TNF) is a key mediator of inflammation, acting as a proinflammatory cytokine with context-dependent dual roles. Here, we describe detailed assays to investigate key downstream signaling pathways of TNF involved in either cell death induction or NF-κB-mediated pro-survival signaling via the TNFR1 axis. We describe steps for target cell and reagent preparation, cell stimulation for cell death induction, and detection of activated intracellular signaling proteins by flow cytometry. For complete details on the use and execution of this protocol, please refer to Unmuth et al.1.