Xf glycolytic rate assay kit

C2C12 cells were seeded at 12,000 cells/well in XFe96 Well plates (Seahorse Bioscience, Billerica, MA, USA). The cells were treated with different concentrations of CCG-1423 and 0.5% DMSO as control, for 24 h. Then, the cells were subjected to extracellular flux analysis using the XF Cell Mito Stress Test Kit (Agilent, 103015-100), XF Glycolytic Rate Assay Kit (Agilent, 103344-100), and XF Real-Time ATP Rate Assay (Agilent, 103592-100). The measurement was performed as previously described [64 (link),65 (link)].

The oxygen consumption rate (OCR) under mitochondrial stress test assay and the proton efflux rate (PER) under glycolytic rate test assay were performed using the Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA). Mitochondrial stress and glycolytic rate test assays were performed using XF Cell Mito Stress Test kit (Agilent Technologies, #103015–100) and XF Glycolytic rate Assay Kit (Agilent Technologies, #103344–100), respectively. The assays were performed according to the manufacturer’s instructions. The H1299 cells (4 × 10⁴ cells/well) were cultured in XF24 cell culture microplate (Agilent Technologies, #102340–100). For mitochondrial stress test assay, oligomycin (1.5 μM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 2 μM) and antimycin A and rotenone mixture (0.5 μM) were added to cell culture plate for determining mitochondrial respiration including basal respiration, maximal respiration and spare respiratory capacity. For glycolytic rate test assay, antimycin A and rotenone mixture (0.5 μM) and 2-deoxy-D-glucose (50 mM) were added to cell culture plate for determining glycolytic flux including basal glycolysis and compensatory glycolysis.

C2C12 cells were seeded in XFe96 Well plates at a density of 1.2 × 10⁴ cells/well (Seahorse Bioscience, Billerica, MA, USA). The cells were then treated with 1.0 m m concentration of valine for 24 h respectively. After valine treatment, Seahorse XFe96 Extracellular Flux Analyzer was used to measure OCR using XF Cell Mito Stress Test Kit (Agilent, Santa Clara, CA, USA; 103015-100) and extracellular acidification rate (ECAR) using XF Glycolytic Rate Assay Kit (Agilent, Santa Clara, CA, USA; 103344-100). For determination of ATP production rate from mitochondria and glycolysis, XF-Real-time ATP Assay Kit (Agilent, Santa Clara, CA, USA; 103592-100). All metabolic assays were performed following the procedure from the manufacturer.