J Vis Exp. 2025 Aug 15;(222). doi: 10.3791/67377.
ABSTRACT
Upon antigen stimulation, naïve T cells undergo rapid proliferation and expansion to effector T cells. Metabolism plays an important role in the generation of biomass needed for these rapidly proliferating cells and for the generation of molecules required for effector T cell differentiation and function, which influence the outcome of the adaptive immune response in infection or cancers. Naïve T cells reprogram their metabolism upon antigenic stimulation to increase the generation of ATP, which is required to support their growth, biosynthesis, and effector functions. ATP can be generated in a cell either by the mitochondrial-oxidative phosphorylation (OXPHOS) pathway or by the glycolytic pathway. Because most of the ATP generated in a dividing, growing cell is used up for the synthesis of proteins, protein synthesis has been used as a surrogate for ATP levels. Protein synthesis can be measured by the incorporation of puromycin, which mimics the 3′ adenosine of a tRNA charged with a modified tyrosine and leads to spontaneous termination of protein translation. Metabolic inhibitors like 2-deoxyglucose (2DG), which blocks the glycolytic pathway, and Oligomycin (O), which blocks complex 5 of the electron transport chain (ETC), can be used to study the dependencies of cellular ATP generation on these two pathways in conjunction with evaluation of protein synthesis in a method called SCENITH. We describe here a variation of this method that detects puromycin incorporation by flow cytometry using chemistry. This method of studying metabolism is relatively easy and can be used for evaluating rare cell populations, as well as patient samples, by flow cytometry.
PMID:40889248 | DOI:10.3791/67377
