Deep metabolic profiling of immune cells by spectral flow cytometry-A comprehensive validation approach.

The advancing field of immunometabolism requires tools that link single-cell metabolism with immune function. Metabolic flow cytometry provides this capability, but its broad adoption has been limited by costly custom reagents and a lack of standardized methods for validating metabolic targets. Here, we present a standardized and user-friendly spectral flow cytometry panel that profiles eight key metabolic pathways at single-cell resolution using only commercially available antibodies, enabling simultaneous analysis of immune phenotype and metabolic activity . Applying this approach to lung myeloid and T cells following intranasal adenoviral CD40L vaccination revealed distinct metabolic phenotypes between resident and infiltrating myeloid cells, as well as functionally divergent metabolic programs in naive, effector, and tissue-resident memory T cells. Additionally, leveraging NAD(P)H autofluorescence allowed label-free detection of glycolysis and expanded the panel's utility. This standardized approach reduces cost and experimental complexity, enabling researchers to elucidate how metabolism drives immune function across broader immunological and clinical contexts.