We used the state-of-the-art Seahorse Extracellular Flux (XF) 96 Analyzer (Seahorse Bioscience, Inc, North Billerica, MA, USA), to measure the oxygen consumption rate (OCR), an indicator of mitochondrial respiration, and the extracellular acidification rate (ECAR), an indicator of glycolysis, in real-time in live intact LCLs.
Several measures of mitochondrial respiration, including basal respiration, ATP-linked respiration, proton leak respiration and reserve capacity, were derived by the sequential addition of pharmacological agents to the respiring cells, as diagramed inFigure 1 . For each parameter, three repeated rates of oxygen consumption are made over an 18 minute period. First, baseline cellular oxygen consumption is measured, from which basal respiration is derived by subtracting non-mitochondrial respiration. Next oligomycin, an inhibitor of complex V, is added, and the resulting OCR is used to derive ATP-linked respiration (by subtracting the oligomycin rate from baseline cellular OCR) and proton leak respiration (by subtracting non-mitochondrial respiration from the oligomycin rate). Next carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP), a protonophore, is added to collapse the inner membrane gradient, driving the ETC to function to its maximal rate, and maximal respiratory capacity is derived by subtracting non-mitochondrial respiration from the FCCP OCR. Lastly, antimycin A, a complex III inhibitor, and rotenone, a complex I inhibitor, are added to shut down ETC function, revealing the non-mitochondrial respiration. The mitochondrial reserve capacity is calculated by subtracting basal respiration from maximal respiratory capacity.
ECAR is primarily a measure of lactate production and can be equated to the glycolytic rate (i.e., glycolysis), and ECAR is measured simultaneously with OCR in the Seahorse assay. Basal ECAR refers to the ECAR measured before the injection of oligomycin. Glycolytic reserve capacity is calculated by subtracting the basal ECAR from the oligomycin-induced ECAR.
One hour prior to the assay, cells were seeded onto poly-D-lysine coated 96-well XF-PS plates at a density of 1.1×105 cells/well in DMEM XF assay media (unbuffered DMEM supplemented with 11 mM glucose, 2 mM L-glutamax, and 1 mM sodium pyruvate). Cells were plated with at least 4 replicate wells for each treatment group. Titrations were performed to determine the optimal concentrations of oligomycin (1.0 µM), FCCP (0.3 µM), antimycin A (0.3 µM) and rotenone (1.0 µM).
Several measures of mitochondrial respiration, including basal respiration, ATP-linked respiration, proton leak respiration and reserve capacity, were derived by the sequential addition of pharmacological agents to the respiring cells, as diagramed in
ECAR is primarily a measure of lactate production and can be equated to the glycolytic rate (i.e., glycolysis), and ECAR is measured simultaneously with OCR in the Seahorse assay. Basal ECAR refers to the ECAR measured before the injection of oligomycin. Glycolytic reserve capacity is calculated by subtracting the basal ECAR from the oligomycin-induced ECAR.
One hour prior to the assay, cells were seeded onto poly-D-lysine coated 96-well XF-PS plates at a density of 1.1×105 cells/well in DMEM XF assay media (unbuffered DMEM supplemented with 11 mM glucose, 2 mM L-glutamax, and 1 mM sodium pyruvate). Cells were plated with at least 4 replicate wells for each treatment group. Titrations were performed to determine the optimal concentrations of oligomycin (1.0 µM), FCCP (0.3 µM), antimycin A (0.3 µM) and rotenone (1.0 µM).
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