Abstract
Metabolic dependences highlight a cell's reliance on specific pathways to meet its bioenergetic needs, with these pathways being interrogated using chemical inhibitors to assess their significance. While surrogate markers of bioenergetics (e.g., oxygen consumption) have yielded important insights, we asked whether metabolic dependences could be defined using ATP as a biomarker. To address this gap, we developed Mitochondrial/Energy Flow Cytometry (MitE-Flo), a method that evaluates the contributions of glycolysis, fatty acid oxidation (FAO), and oxidative phosphorylation (OXPHOS) to cellular ATP content. In models of mitochondrial disease due to complex I or complex IV deficiency, we identified impaired OXPHOS with a compensatory shift to glycolysis. To define the utility of ATP monitoring in immunometabolism research, we analyzed previously inaccessible cell populations: light zone (LZ) and dark zone (DZ) germinal center (GC) B cells. Highly proliferative DZ B cells exhibited elevated ATP levels and a preference for FAO and OXPHOS over glycolysis, with uniform increased activity across ETC complexes. In contrast, less proliferative LZ B cells showed lower ATP levels and an equal reliance on glycolysis and OXPHOS. Using ATP as a biomarker to define metabolic dependences provides valuable insights into disease states and elusive immune cell subtypes, thereby enhancing the metabolism research toolkit.