Seahorse XF Analysis of Glycolysis and Mitochondrial Respiration

Seahorse XF technology (Agilent Technologies, Santa Clara, CA) was used to monitor extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) to measure levels of glycolysis and mitochondrial respiration, respectively. CHOK1-GLP-1R cells were seeded in Seahorse XF96 cell culture microplates at a concentration of 20,000 cells per well and incubated at 37°C overnight. On the experimental day, cells were washed two times with 200 µl of assay media [complete media without sodium bicarbonate, FBS, or Geneticin G418 Sulfate; pH = 7.38–7.42; 37°C] and then incubated in assay media with or without the GLP-1R antagonist exendin-9 (Ex9, 500 nM) for 80 min in a carbon dioxide-free incubator. ECAR and OCR measurements were made at 7-min intervals before (Baseline) and after injection of GLP-1RA (20 pM) ± OEA (10 µM) from the Seahorse XF96 sensor cartridge. To verify that the observed changes in ECAR were due to changes in glycolysis, the same experiment was performed but using the glycolytic inhibitor 2-deoxyglucose (2-DG; 20 mM) instead of Ex9 during the 80-min preincubation. All reagents were brought to a pH of 7.38–7.42 at 37°C before use.