4.11. Seahorse Agilent Seahorse XF Cell Mito Stress Test

To evaluate the changes in mitochondrial function and cell metabolism, cells underwent Seahorse Agilent Seahorse XFe Cell Mito Stress test (Agilent, Santa Clara, CA, USA) to measure changes in mitochondrial function via oxygen consumption rate (OCR). Agilent Seahorse XFe cell mito stress test was performed according to the manufacturer’s instructions. In brief, cells were seeded at the optimized cell density for each of the different cell lines in Seahorse XFe96 microplates and incubated at 37 °C/5% CO₂ for 24 h. On the day of assay, the cell culture growth medium in the cell culture microplate was replaced with pre-warmed (37 °C) assay medium (XF DMEM, 1 mM Pyruvate, 2 mM Glutamine, and 10 mM glucose). The cell culture microplate was incubated in a non-CO2 incubator at 37 °C for 1 h prior to the assay to allow media temperature and pH to reach equilibrium. The modulating agents (oligomycin, FCCP, and antimycin A + rotenone) were prepared in assay medium and injected into the injection ports. The OCR was measured and analyzed using the Seahorse XFe Mito Stress Test Report Generator. At the end of the assay, cells were stained with crystal violet. The number of stained cells was counted and used for normalization.