Protocol for measuring the caspase-8 activity at the DED filaments in adherent cells

The key stage of extrinsic apoptosis is the activation of procaspase-8 at the death-inducing signaling complex (DISC), where procaspase-8 assembles into death effector domain (DED) filaments. Here, we present a protocol to measure caspase-8 activity directly at the DISC. We describe steps for cell culture, apoptosis induction, immunoprecipitation, caspase-8 assay, and western blot analysis. This approach enables the analysis of caspase-8 activation in its native complex and can be applied to assess the efficacy of pharmacological inhibitors targeting caspase-8. For complete details on the use and execution of this protocol, please refer to König et al.¹.