Fig. 9

Fig. 9

Methods to measure EC metabolism using radioactive tracers and Seahorse XF analyzer. a Schematic representation of glycolytic flux measurements with [5–³H]-glucose. A single tritium present on 5C glucose is released as water in the ninth step of glycolysis catalyzed by enolase. b Schematic representation of the modified Glycolysis Stress Test. The first measurements of ECAR are performed while ECs are incubated in glycolysis stress test medium (without glucose and pyruvate). The injection of glucose leads to the saturation of glucose concentration and allows measuring glycolytic rate (blue). Second, injection of oligomycin blocks oxidative ATP production and shifts the energy production to glycolysis, with the subsequent increase in ECAR revealing the cellular maximum glycolytic capacity (green). The difference between glycolytic capacity and glycolysis rate defines glycolytic reserve. The final injection of 2-deoxyl-glucose (2-DG) inhibits glycolysis, and the resulting decrease in ECAR confirms that the ECAR produced in the experiment is due to glycolysis. ECAR, prior to glucose injection or after 2-DG injection, is referred to as non-glycolytic acidification (pink). c Schematic representation of the modified Seahorse Cell Mito Stress Test. First injection of oligomycin blocks ATP synthase and allows the calculation of the ATP coupled oxygen consumption rate (OCR_ATP; red). Second, injection of FCCP maximizes the OCR by uncoupling the OXPHOS, enabling to calculate the spare respiration (reserve capacity). Third, antimycin-A treatment blocks complex III of ETC enables the calculation of the basal mitochondrial respiration (OCR_BAS; green), the maximal mitochondrial OCR (OCR_MAX; orange), the proton leakage (blue), and the non-mitochondrial OCR (pink)