ATP-Red enables single-cell bioenergetic phenotyping by flow cytometry.

Most established bioenergetic assays lack single-cell resolution and may mask metabolic heterogeneity. The main flow-cytometry-based approach for bioenergetic analysis infers energetic state from protein synthesis, although translation can become uncoupled from ATP availability under physiologic and pathologic conditions. Here, we evaluated ATP-Red as a flow-cytometry-compatible readout of energetic state at single-cell resolution. We benchmarked ATP-Red against orthogonal approaches, including colorimetric ATP quantification, the ATP/ADP biosensor PercevalHR, and extracellular flux analysis, across ATPase inhibition, glycolytic and oxidative blockade, mitochondrial dysfunction, and immune activation. Across these settings, ATP-Red tracked biologically meaningful energetic changes as a relative ATP-linked fluorescence readout. When combined with immunophenotyping, ATP-Red also captured bioenergetic heterogeneity and pathway use in T cells following activation and during influenza virus infection. These findings support ATP-Red as a practical and scalable approach for single-cell bioenergetic phenotyping.