Glycolysis Stress test with the Seahorse Extracellular XFe Flux Analyser

The glycolysis levels of the cells were measured using the Seahorse Extracellular XF^e Flux Analyser as reported previously and according to the manufacturer's instructions [80]. Briefly 40,000 cells/well were seeded in a Seahorse XF24 Cell Culture Microplate. The Seahorse Base medium included 1 mM glutamine, was adjusted to pH 7.35 and was kept at 37°C after filtration. The normal media of the cells was removed and Glycolysis media was added before the plate was left in a CO₂-free incubator for 1 h. All the injections prepared meanwhile were loaded on the injection plate and both plates were inserted and the experiment followed the suggested protocol. After the experiment, a protein quantification assay was performed and the results were normalized to protein concentration/well. The glycolysis levels are presented as x-fold increase in glycolysis rates measured in mpH/min (ECAR) from three independent experiments performed in triplicate.