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

## TL;DR

This paper provides a detailed protocol for measuring caspase-8 activity at the DISC in adherent cells, enabling analysis of apoptosis and drug efficacy.

## Contribution

A novel protocol combining immunoprecipitation with caspase-8 activity assays directly at the DISC is introduced.

## Key findings

- The protocol allows measuring caspase-8 activation in its native complex.
- It can assess the efficacy of pharmacological inhibitors targeting caspase-8.

## Abstract

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

•Steps for measuring caspase-8 activity at the death-inducing signaling complex (DISC)•Instructions for combining immunoprecipitation (IP) with a caspase-8 activity assay•Analysis of IP efficiency and caspase-8 enzymatic activity from the same sample

Steps for measuring caspase-8 activity at the death-inducing signaling complex (DISC)

Instructions for combining immunoprecipitation (IP) with a caspase-8 activity assay

Analysis of IP efficiency and caspase-8 enzymatic activity from the same sample

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.