Cellular Respiratory Profiling of MEFs

ECAR and OCR measurement were performed using a Seahorse XFe24 analyzer (Seahorse Bioscience) on MEFs (20,000 cells/well) plated on XFe24 tissue culture plates coated with fibronectin (3 μg/ml). Seahorse XF Cell Mito Stress Test Kit (103015-100), XF Glycolytic Rate Assay Kit (103344-100), XF Real-Time ATP Rate Assay Kit (103592-100), and XF Palmitate Oxidation Stress Test Kit (103693-100) were used according to the manufacturer’s protocol. For OXPHOS experiments testing glucose, glutamine, and fatty acid as respiratory substrate [44 (link)], cells were incubated with DMEM minimum medium (without glucose, sodium pyruvate, L-Glutamine, and 1% FBS) 24 h before the assay. One hour prior to measurements, cells were treated/untreated, respectively, with UK5099 (10 µM, Selleckchem, S5317), BPTES (20 µM, Selleckchem, S7753), or Etomoxir (100 µM, Selleckchem, S8244), and incubated (CO₂-free atmosphere) at 37 °C.