2.10. Seahorse XF glycolysis stress assay

For the Seahorse XF glycolysis stress assay, the extracellular acidification rate (ECAR) was measured using the Seahorse XF Glycolysis Stress Test Kit from Agilent (#103015–100; Agilent Technologies) and the XF24 Extracellular Flux Analyzer, following a previously described protocol. In brief, cells were seeded at a density of 5 × 10⁴ cells per well in Seahorse XF24 V7 PS Cell Culture Microplates and cultured overnight. ECAR was measured in XF base medium supplemented with 2 mM glutamine (pH 7.4) following the sequential injection of glucose (10 mM), oligomycin (1 μM), and 2‐DG (50 mM). The following calculations were performed:

Glycolysis: Maximum rate measurement before oligomycin injection minus the last rate measurement before glucose injection, representing the basal glycolysis level.

Glycolytic Capacity: Maximum rate measurement after oligomycin injection minus the last rate measurement before glucose injection, representing the maximum glycolysis level.