Extracellular Flux Assays for the Measurement of Glycolysis and Mitochondrial Respiration in Brain Cancer Cells

Several essential biological processes, such as cell growth, survival, and death, are regulated by cellular metabolism. Both normal and cancer cells exhibit metabolic flexibility to adjust to changes in the energy demand in response to alterations in their external microenvironment. This metabolic plasticity is more pronounced in cancer cells, which often reprogram their metabolism to promote their growth and survival. Extracellular flux analysis is a powerful and widely used technique to study cellular metabolism in real time. This approach provides dynamic and functional insights into two key bioenergetic pathways: glycolysis and oxidative phosphorylation (OXPHOS), under both normal and stressed conditions in live cells. In this chapter, we discuss different extracellular flux assays for assessing glycolytic activity, including the glycolysis stress test (GST) and glycolytic rate assay (GRA), as well as OXPHOS using cell mito stress test (MST) with the Agilent Seahorse extracellular flux analyzer. We also highlight key factors to consider for successfully conducting extracellular flux assays.