Figure 1
From: Oxidative stress induces mitochondrial dysfunction in a subset of autistic lymphoblastoid cell lines
The Seahorse assay. Oxygen consumption rate is measured before and after adding pharmacological agents to respiring cells. Measurement of oxygen consumption over 6 min is made repeatedly. Three measurements are made and averaged to provide reliable measurements. For the first 18 min, total cellular oxygen consumption is measured. Basal respiration can be calculated from this quantity by subtracting non-mitochondrial respiration. Next oligomycin, an inhibitor of adenosine-5’-triphosphate (ATP) respiration, is added and a measurement of this is made over the next 18 min. This quantity can be subtracted from the total cellular oxygen consumption to determine ATP-linked respiration and non-mitochondrial respiration can be subtracted from this quantity to obtain proton-leak respiration. Next carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon, a protonophore, is added and a measurement of this is made over the next 18 min. The protonophore collapses the inner membrane gradient by making the inner membrane permeable to protons. This drives the electron transport chain to function at its maximum rate. Subtracting non-mitochondrial respiration from this quantity produces a measure of maximum respiratory capacity. Finally antimycin A, a complex III inhibitor, and rotenone, a complex I inhibitor, are added to shut down electron transport chain function. The resulting measurement over the next 18 min represents non-mitochondrial respiration, a measurement that can be used with the other measurements to calculate respiratory parameters. Finally, reserve capacity is calculated by subtracting basal respiration from maximum respiratory capacity.
