Mitochondrial respiration and glycolysis with Seahorse bioanalyzer

Cell Mito Stress Tests and the Glycolytic Rate Assays were performed using a Seahorse XFe24 Analyzer (Agilent, Santa Clara, CA). An equal number of cells (20,000 ASCs/well) were seeded in Seahorse cell culture microplates (Agilent, Santa Clara, CA) 1 day prior to the experiments and incubated in MesenPro™ growth media at 37 °C and 5% CO₂ overnight. The sensor cartridges were hydrated in Seahorse XF Calibrant Solution (Agilent, Santa Clara, CA) overnight and incubated in a non-CO₂, 37 °C incubator 1 day before the experiment. XF Assay media was prepared according to the manufacturer’s instructions containing Agilent Seahorse XF Base Medium, 10.5 mM glucose, 1 mM sodium pyruvate, 2 mM l-glutamine, and 5 mM HEPES, and the pH was adjusted to 7.4. An automated protocol for the Cell Mito Stress Test used serial injections of inhibitors and uncouplers to determine the oxygen consumption rate (OCR) in each respiratory state. After a period of equilibration, the basal OCR was determined. Then, 1.0 μM oligomycin, an ATP synthase inhibitor, was added to determine leak respiration that is not coupled to the ATP synthesis. Afterwards, 1.0 μM FCCP was added to determine the maximal respiration of the electron transport chain. Finally, 0.5 μM rotenone/antimycin A was added to inhibit complexes I and II, respectively, to determine the residual respiration indicating proton leak in the mitochondria after inhibition of the electron transport chain.

An automated Glycolytic Rate Assay utilized both the OCR and the extracellular acidification rate (ECAR) to calculate the glycolytic proton efflux rate (PER). After the basal glycolytic PER was measured, the compensatory glycolysis was measured by inhibiting oxidative phosphorylation by injecting the complex I and II inhibitors rotenone and antimycin A (0.5 μM final concentration). Afterwards, 2-deoxy-d-glucose (50 mM final concentration) was added to inhibit hexokinase, thereby inhibiting glycolysis to determine the residual acidification. After glycolytic PER was determined for all states, the residual acidification values were subtracted from basal and compensatory glycolysis to account for acidification from other sources such as the TCA cycle.