Abstract
Cellular metabolism dictates immune cell function, yet we lack tools to functionally profile immunometabolism in low-yield, complex samples. We present a flow cytometry-based protocol for measuring cellular energetics through noncanonical amino acid tagging (CENCAT) in human peripheral blood and murine tissue immune cells. We describe steps for sample preparation, metabolic inhibition, protein synthesis analysis using click chemistry, immunophenotyping, and calculation of metabolic dependencies. For complete details on the use and execution of this protocol, please refer to Vrieling et al.1.