2.12. Seahorse experiments

Functional mitochondrial measurements were performed using the Seahorse XF Cell Mito Stress Test according to manufacturer's instructions. Briefly, AML12 cells were seeded onto an XF24 Cell Culture Microplate (Agilent Seahorse Bioscience) at 3,000 cells/well and incubated at 37 °C overnight in regular media. The following morning, regular media was replaced for Seahorse XF Base Medium containing 1 mM pyruvate (Gibco, 11360070), 2 mM l-glutamine (Gibco, 25030149) and 25 mM glucose (Sigma, G8270). Cell metabolic rates were measured using an XF24 Extracellular Flux Analyzer (Agilent Seahorse Bioscience) following the sequential addition of 1 μM oligomycin (oligo; Sigma 75351; port A), 4 μM carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP; Sigma, C2920; Port B) and 0.5 μM antimycin A (AA; Sigma, A8674; Port C) + 0.5 μM rotenone (rot; Sigma, R8875; Port C). Oxygen consumption rate (OCR) was normalized to protein concentration using the Protein Assay reagent (Bio-Rad, 500-0006) and expressed as % from baseline. Non-glycolysis acidification (NGA), glycolysis (G), glycolysis capacity (GC) and glycolytic reserve (GR), were also evaluated using the Seahorse XF Glycolysis Stress Test according to manufacturer's instructions. Briefly, AML12 cells were seeded onto an XF24 Cell Culture Microplate (Agilent Seahorse Bioscience) at 3,000 cells/well and incubated at 37 °C overnight in regular media. The following morning, regular media was replaced for Seahorse XF Base Medium containing 1 mM l-glutamine (Sigma), and cells were incubated for one hour at 37 °C in a non-CO₂ incubator. Extracellular acidification rate (ECAR) was measured following the sequential addition of 10 mM glucose (Sigma, G8270; port A), 1 μM oligomycin (oligo; Sigma, 75351; port B), and 50 mM 2-DG (Sigma, D6134; port C).