Cellular Metabolic Profiling using Seahorse

Respiratory capacity and glycolytic activity of cells were performed with the Seahorse XF24e Extracellular Flux Analyser (Seahorse Bioscience, Billerica, MA, USA) on attached cells as described^{9 (link)}. Briefly, 2 × 10⁴ cells/well were seeded in XF24 V7 microplates for 24 h before drug treatment. To determine oxygen consumption rate (OCR), cells were resuspended in DMEM (D5030, Sigma-Aldrich) with l-glutamine (2 mM) and d-glucose (10 mM) and the following drugs were added: 1 µM oligomycin, 0.25–0.5 µM FCCP, 1 µM rotenone, and 1 µM antimycin A. To determine extracellular acidification rate (ECAR), cells were resuspended in glucose-free DMEM (D5030, Sigma-Aldrich) with l-glutamine (2 mM) and the following compounds were added: d-glucose (10 mM), oligomycin (1 µM), and 2DG (10 mM).