2.6.2. Oxygen Consumption (OCR) and Extracellular Acidification Rate (ECAR) Measurements

Extracellular flux measurements were performed using a seahorse XF96 analyzer (Agilent, Santa Clara, CA, USA) as described by the manufacturer. Briefly, the oxygen consumption rate (OCR) and Extra Cellular Acidification Rate (ECAR) were measured using the Seahorse XF96 analyzer (Seahorse Bioscience, Agilent, US) at 37 °C. For AML cell lines, 1 × 10⁵ viable cells (evaluated by DAPI-using flow cytometry measurements) were seeded per well in poly-L-lysine (Sigma-Aldrich) coated (incubation at room temperature for 20 min) Seahorse XF96 plates in 180 μL XF Assay Medium (Modified DMEM, Seahorse Bioscience), respectively, in the presence of 2 mM of Glutamine and NaOH to adjust the final pH. For OCR measurements, XF Assay Medium was supplemented with 10 mM Glucose and 2.5 µM oligomycin A (Port A), 2.5 µM FCCP (carbonyl cyanide-4-(trifluorometh oxy) phenylhydrazone) (Port B), and 2 µM antimycin A, together with 2 µM Rotenone (Port C), were sequentially injected in 20 µL volume to measure basal and maximal OCR levels (all reagents from Sigma-Aldrich). For ECAR measurements, Glucose-free XF Assay medium was added to the cells in addition to 10 mM Glucose (Port A), 2.5 µM oligomycin A (Port B), and 100 mM 2-deoxy-D-glucose (Port C) (all reagents from Sigma-Aldrich). All XF96 protocols consisted of 4 times mix (2 min) and measurement (2 min) cycles, allowing for determination of OCR at basal and in between injections. Both basal and maximal OCR levels were calculated by assessing metabolic response of the cells in accordance with the manufacturer’s suggestions. The OCR measurements were normalized to the viable number of cells used for the assay.