Abstract Text: T cell activation and differentiation depends on dynamic changes in essential metabolic pathways such as glycolysis, oxidative phosphorylation and fatty acid metabolism. A deeper understanding of the metabolic regulation of immune cell function is needed for the improvement of immunotherapy approaches. Methods are needed to interrogate metabolic pathways at single-cell resolution within a heterogeneous population of rare cell sub-types. Puromycin incorporation into nascent polypeptides has been used for metabolic profiling by flow cytometry (Scenith method [1]). However, this method is time consuming, requiring cell fixation and permeabilization. Additionally, this method does not provide a direct measurement of available cellular ATP. Puromycin can stimulate polypeptide synthesis [2] and prevent insulin-stimulated increase of glycolysis [3]. Puromycin labeling is not reliable across cell types and does not accurately measure translation rates in energetically challenged cells [4]. We have developed a direct and simplified approach to measure ATP levels in live cells by flow cytometry, allowing for the discrimination of rare sub-populations while interrogating important cellular metabolic pathways, such as glycolysis and oxidative phosphorylation. This method is rapid – results can be obtained in < 1hour (compared to > 2 hours with puromycin) and yields an accurate measurement of cellular ATP production and its dependency on metabolic pathways of interest. Using primary human and murine leukocytes, we show that this method accurately depicts the metabolic changes occurring during T cell activation, differentiation and memory formation. This method will facilitate research of cellular metabolic pathways in many cell types across species and fields of study.
