Protocol for metabolic profiling of antigen-specific CD8⁺ T cells using spectral flow cytometry

CD8⁺ T cells rely on tightly regulated metabolic remodeling to support effector function. Here, we present a protocol for single-cell metabolic profiling of rare antigen-specific CD8⁺ T cells in unstimulated and antigen-stimulated conditions using spectral flow cytometry. The workflow enables detailed assessment of key metabolic pathways, including glycolysis, fatty acid oxidation, amino acid metabolism, the pentose phosphate pathway, and mitochondrial oxidative phosphorylation. For complete details on the use and execution of this protocol, please refer to Mülling et al.¹.