2.6. Seahorse XF Analyzer Respiratory Assay

Cells (6 × 10⁴ cells per well) were seeded in Seahorse XFe96 FluxPaks for metabolic analysis with an extracellular flux analyzer XF96 (Seahorse, Agilent, Santa Clara, CA, USA). The sensor cartridge was hydrated in a 37 °C non-CO₂ incubator one day before the experiment. Cells were serum-starved overnight, treated with LPA at the indicated concentrations for the given time periods, washed, and incubated with the appropriate assay medium for 1 h in a 37 °C non-CO₂ incubator according to the manufacturer’s instructions. Cellular oxygen consumption rate (OCR) was determined using the XF Cell Mito Stress Test (Agilent). Optimized stressor concentrations were added as follows: 2 μM oligomycin (complex V inhibitor), 1.75 μM cyanide p-trifluoromethoxy-phenylhydrazone (FCCP; proton gradient disruption), and 2.5 μM antimycin A (inhibitor of complex I and III). OCR was normalized to protein concentrations, and data from 3 independent experiments are shown.