Measurement of oxygen consumption in AML cultured cells using Seahorse assay

All XF assays were performed using the XFe24 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA). The day before the assay, the sensor cartridge was placed into the calibration buffer medium supplied by Seahorse Biosciences to hydrate overnight. Wells of Seahorse XFe24 microplates were coated with 50 µl of Cell-Tak (Corning; Cat#354240) solution at a concentration of 22.4 µg/ml and kept at 4 °C overnight. Then, Cell-Tak-coated Seahorse microplates were rinsed with distilled water and AML cells were plated at a density of 10⁵ cells/well with XF base minimal DMEM media containing 11 mM glucose, 1 mM pyruvate, and 2 mM glutamine. Then, 180 µl of XF base minimal DMEM medium was placed into each well and the microplate was centrifuged at 80g for 5 min. After 1 h incubation at 37 °C in CO₂-free atmosphere, basal oxygen consumption rate (OCR, as a mitochondrial respiration indicator), and extracellular acidification rate (ECAR, as a glycolysis indicator) were performed using the Seahorse XFe24, and analyzed using Wave software (version 2.6.1).