Integrated Functional and Metabolomic Profiling of Melanoma Cells Treated with Redox-Active Compounds

Metabolic plasticity is a hallmark of melanoma progression and therapy resistance. This chapter describes a reproducible protocol for comprehensive metabolic profiling of melanoma cells exposed to redox-active compounds such as emodin and aloe-emodin. The method integrates cell proliferation assays, mitochondrial functional analysis using Seahorse extracellular flux measurements, reactive oxygen species (ROS) quantification, LC-MS-based metabolomics, stable-isotope tracing, and ¹³C metabolic flux analysis (¹³C-MFA). The workflow enables quantitative characterization of treatment-induced alterations in energy metabolism, redox balance, and central carbon fluxes in melanoma cell lines with distinct metabolic phenotypes. The procedures are presented in a step-by-step format suitable for researchers unfamiliar with the individual techniques and can be adapted to other cancer models or metabolic perturbations.