Mitochondrial Bioenergetics Profiling

To assess mitochondrial function, a Mito stress test on a Seahorse XFp analyzer (Agilent, Santa Clara, CA, USA) was performed. The entire culture media was replaced with Seahorse XFp Base medium (pH 7.4). Following the manufacturer’s protocol, the mitochondrial inhibitors oligomycin, FCCP (trifluoromethoxy carbonyl cyanide phenylhydrazone), and rotenone/antimycin A were consecutively injected into the ports of the cartridge. Injection of the first chemical, known as an ATP synthase inhibitor, determines the production of ATP. FCCP makes the inner mitochondrial membrane permeable to protons and allows maximum electron flux through the electron transport chain (ETC). Thus, collapse of the inner membrane gradient is induced, driving mitochondria to respire at the maximum rate. The latter of the chemicals directly inhibits Complex I of the ETC and shuts mitochondrial respiration down (Figure 1). Such a combination of inhibitors allows for the assessment of several parameters: basal and maximal respiration, ATP levels, and spare respiratory capacity, as well as other mitochondrial characteristics such as proton leak, and non-mitochondrial respiration (Figure 2). For each experiment, the oxygen consumption rate (OCR) was measured, and the data were analyzed using Seahorse Wave Desktop Software v2.6.0 (Agilent, Santa Clara, CA, USA).