Oxygen Consumption Rate Analysis in Cell Models

Oxygen consumption rate (OCR) was measured in KO cell models and CLN5 cultured fibroblasts (together with their respective controls), using an XFe24 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent, Santa Clara, CA). Cells were plated in XF 24-well cell culture microplates at a density of 6E + 04 cells/well and 5E + 04 cells/well for cell lines and primary fibroblasts, respectively. Measurements of endogenous respiration were performed with non-buffered DMEM medium supplemented with 1 mM pyruvate, 2 mM glutamine and 10 mM glucose. After baseline measurements, OCR was analyzed by the sequential injection of 1 mM of oligomycin, 2 mM of carbonyl-cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and 0.5 mM of rotenone plus antimycin A (all chemical were from Sigma Aldrich, St. Louis, MO). Data were expressed as pmol of O₂/min normalized post-assay by the fluorescence CyQUANT Cell Proliferation Assays (Invitrogen™, Carlsbad, CA), as reported elsewhere^{55 (link)}.