2 deoxyglucose

The Seahorse XF96 Extracellular Flux Analyzer (Agilent) was used to assess OCR and ECAR. Cells were seeded in poly-l-lysin-coated XF96 cell culture microplates (Agilent) at the density of 15,000 cells per well and cultivated overnight in the standard growth medium. Next day, the growth medium was replaced by the assay medium (DMEM with 2 mM glutamine but without glucose, carbonate and a pH indicator dye, pH 7.4 at 37 °C). For OCR measurements, the assay medium was additionally supplemented with pyruvate (1 mM), glucose (10 mM) and fatty acids-free bovine serum albumin (0.2% w/v). The plates were incubated in a 37 °C incubator without CO₂ for 1 h, followed by the measurement in the Seahorse instrument. For OCR, the wells were consecutively injected with oligomycin (1 µM f.c.), carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 1 µM f.c.) and rotenone/antimycin A (both 0.5 µM f.c.). For ECAR measurements, glucose (10 mM f.c.) was injected first, followed by oligomycin (1 µM f.c.; Cayman Chemicals, Ann Arbor, MI, USA) and then 2-deoxyglucose (50 mM f.c.; Cayman Chemicals). Once measurements were completed, the cell nuclei were stained by Hoechst 33342 and the number of cells per well was determined using an imaging plate reader (Molecular Devices, San Jose, CA, USA). Measured extracellular flux rates were normalized to the cell number.