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 • Step-by-step workflow for reproducible spectral flow metabolic profiling • Stimulation of antigen-specific CD8 + T cells derived from blood or spleen • Optimized antibody panels quantify key metabolic enzymes in resting/active states • Guidance on analysis and interpretation of metabolic pathway activity in T cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
Graaf et al. (Wed,) studied this question.