Rotenone plus antimycin a

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)}.

The XF96 extracellular flux analyzer (Seahorse Bioscience) was used to measure the glycolysis and mitochondrial respiration in BCSCs as described previously [50 (link)]. Briefly, cells were seeded in the 96-well culture plate (Seahorse Bioscience) at a density of 20,000 cells/well overnight. For the mitochondrial stress test, cells were incubated with XF base medium (pH 7.4) supplemented with 5.5 mM glucose, 2 mM glutamine and 1mM sodium pyruvate for 1 h at 37 °C in a non-CO₂ incubator. Oxygen consumption rate (OCR) was then measured by sequential injection of Oligomycin A (Sigma, 75351, St. Louis, MO, USA), FCCP (Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) (Sigma, C2920), and Rotenone plus Antimycin A (Sigma, R8875 and A8674). For the glycolysis stress test, cells were incubated with XF base medium (pH 7.4) supplemented with 2mM glutamine for 1 h at 37 °C in a non-CO₂ incubator. ECAR (Extracellular acidification rate) was measured by consecutive injection of d-Glucose, Oligomycin A, and 2-deoxy-d-glucose. Assay well cell counts were measured using Cyquant Cell Proliferation Assay (Invitrogen, C7026, Waltham, MA, USA to normalize the OCR and ECAR values in each well. The Seahorse XF cell Mito stress test and glycolysis stress test report generators were used to analyse the data.