Mitochondrial Bioenergetics Profiling using Seahorse Assay

Seahorse assays were conducted in accordance with a published protocol (50 (link)). Mitochondrial bioenergetic measurements were performed using the Seahorse XFe96 Extracellular Flow Analyzer (Agilent Technologies, Santa Clara, CA, USA) to measure the oxygen consumption rate (OCR) in different respiratory states. 2.5 x 10⁴ LS174T cells in 80 μL medium were seeded in XF96 Cell Culture microplate and cultured overnight. Then cell culture media were replaced with Seahorse XF modified media before cells were treated. In the standard assay, cells were treated sequentially with 1 μM oligomycin, 1.0 μM FCCP, and a mixture of 1.0 μM rotenone and 1.0 μM antimycin A. To determine if compounds inhibited ETC complex V/ATP synthase, oligomycin was replaced with DMSO, or compounds dissolved in DMSO. To test whether compounds were ETC complex I or III inhibitors, rotenone and antimycin A were replaced with DMSO, or compounds in DMSO solution.