Figure EV1. Metabolic reactions that generate net pH changes.

1. The main driver of extracellular acidification in most cell culture monolayer systems is lactate efflux. Uncharged glucose is taken up by the cell and converted to anionic pyruvate via glycolysis. Subsequent fermentation to lactate and efflux via monocarboxylate transporters causes a net pH change in the experimental medium that is detected by the XF Analyzer. GLUT, glucose transporter; MCT, monocarboxylate transporter; PYR., pyruvate; LAC., lactate.
2. Other reactions can cause a net pH change in the experimental medium, such as the efflux of glucose‐derived carbon as anionic pyruvate or CO₂ evolved from the oxidative pentose phosphate pathway (PPP) that ultimately forms bicarbonate and H⁺.
3. CO₂ evolution by mitochondrial dehydrogenases also generates bicarbonate that can be detected by ECAR. CO₂ is evolved from various mitochondrial enzymes including isocitrate dehydrogenase (IDH) and the three oxoacid dehydrogenases: pyruvate dehydrogenase (PDH), α‐ketoglutarate dehydrogenase (α‐KGDH), and the branched chain keto acid dehydrogenase (BCKDH). α‐KIC, α‐ketoisocaproic acid, the keto acid derivative of leucine.