Glycolytic Profiling of Mouse Embryonic Stem Cells (mESCs)

Mouse embryonic stem cells (mESCs) can be captured in vitro in different pluripotency states through media modulation, mimicking their natural environment during early embryo development. As highly proliferative cells, mESCs prefer to use glycolysis to support the energetic and biosynthetic demands, even in the presence of oxygen. Indeed, glycolysis can not only supply ATP at a much faster rate, when compared to other catabolic pathways, but also provides biosynthetic substrates to meet anabolic requirements. Considering that ESCs cultured in different media conditions display distinct metabolic requirements, it is of utmost importance to have a robust metabolic characterization methodology to understand how subtle metabolic variations may be coupled to ESC identity. Here we describe how to profile the glycolytic activity of naive mouse ESC, using the established Seahorse XFe24 Live-cell Metabolic Assay. This may be a useful protocol for understanding how the glycolytic function of mESCs changes in certain circumstances and how is it coupled to diverse pluripotency/differentiation phenotypes.