Abstract
Ferroptosis is a regulated form of cell death caused by the excessive accumulation of iron-dependent lipid peroxidation. It has been implicated in various pathological processes and diseases, and its modulation involves multiple proteins associated with iron and lipid metabolism. To better understand these mechanisms and monitor the ferroptosis process, there is a need for reliable and high-throughput methods to evaluate variations in protein expression levels. In-Cell Western assays provide a simple and rapid assay method for detecting biomarkers and signaling proteins in whole cells using antibodies. This assay involves seeding cells in microtiter plates, followed by fixation/permeabilization and subsequent labeling with primary antibodies and infrared-conjugated secondary antibodies. In this chapter, we introduce the protocol for the In-Cell Western assay for detecting intracellular proteins during ferroptosis.