Agilent seahorse xf calibrant

Seahorse XFp analyzer (XFp, Seahorse Biosciences, MA) was used to evaluate the mitochondria oxygen consumption rate (OCR) of C2C12 myotubes. After C2C12 myoblasts were seeded and differentiated in Seahorse XFp miniplates, myotubes were pretreated with AN07 (0.1 or 1 μM) for 1 h followed by the addition of LPS (100 ng/mL) for 24 h. Before assay, the fluorescence probes were activated by Agilent Seahorse XF calibrant at 37 °C in a non-CO2 incubator overnight. Subsequently, the culture medium was replaced with Agilent Seahorse XF base medium (with 1 mM pyruvate, 2 mM glutamine, 10 mM glucose, and pH adjusted to 7.4), and the miniplates were then placed in a non-CO₂ incubator at 37 °C for 1 h. The key parameters of mitochondrial function were measured using Seahorse XFp Cell Mito Stress Test, which were obtained at the baseline and following sequential injection of oligomycin (1 μM), FCCP (1 μM), and a mixture of antimycin A plus rotenone (AA/ROT, 1 μM).

The Agilent Seahorse XF technology was used to analyze the cell energy phenotype of HA and A172 cells using the Agilent Seahorse XFp Cell Energy Phenotype Test Kit (Agilent, 103275-100, Santa Clara, CA, USA) based on the instructions. HA and A172 cells were grown in the Agilent Seahorse XFp Cell Culture Miniplate and cultured overnight. The Agilent Seahorse XFp Sensor Cartridge was hydrated using Agilent Seahorse XF Calibrant at 37 °C in a non-CO₂ incubator overnight. The Agilent Seahorse Sensor Cartridge, Calibrant, and Miniplate were obtained from the Agilent Seahorse XFp FluxPak (Agilent, 103022-100). The assay medium was prepared by supplementing Agilent Seahorse XF Base Medium (Agilent, 102353-100) with 1 mM pyruvate (Sigma, S8636, St. Louis, MO, USA), 2 mM glutamine (Sigma, G8540), and 10 mM glucose (Sigma, G8769). The cell culture medium of HA and A172 were replaced with the assay medium and cultured in a non-CO₂ incubator for 1 h. Oligomycin and cyanide p-trifluoromethoxylphenyl-hydrazone (FCCP) from the Agilent Seahorse XFp Cell Energy Phenotype Test Kit were combined to develop a stressor mix loaded into every port A of the hydrated sensor cartridge. The Agilent Seahorse XF Cell Energy Phenotype test was run using the Agilent Seahorse XFp Analyzer (Agilent,102745-100). The data were analyzed with the Agilent Seahorse XF Cell Energy Phenotype Test Report Generator.