Mitochondrial bioenergetics measurements

Mitochondrial bioenergetic measurements were performed using a Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) to measure the oxygen consumption rates (OCR) during various states of respiration. The OCR were measured in the presence of different substrates, inhibitors, and uncouplers of the electron transport chain using previous methods with slight modifications.^45–48 The stocks used for the assays were 500 mM pyruvate, 250 mM malate, and 30 mM ADP, and 1 M succinate (pH for all was adjusted to 7.2). Assay solutions of 1 mM oligomycin A, 1 mM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and 1 mM rotenone were prepared in ethanol.

As per the instructions from the XFe24 Extracellular Flux kit, the sensor cartridge was hydrated and kept at 37°C overnight before the experiment. The injection ports A to D of the sensor cartridge were loaded with 75 μL of different combinations of the above substrates/inhibitors/uncouplers as follows: Before loading, the stocks were diluted appropriately in the respiration buffer (RB) (125 mM KCl [potassium chloride], 0.1% BSA, 20 mM HEPES, 2 mM magnesium chloride [MgCl₂], and 2.5 mM monobasic potassium phosphate [KH₂PO₄]; pH 7.2) to get the final concentrations in the respiration chamber of 5 mM pyruvate, 2.5 mM malate, and 1 mM ADP (via port A), 1 μM oligomycin A (via port B), 4 μM FCCP (via port C), and 0.1 μM rotenone and 10 mM of succinate (via port D) starting with the initial volume of 525 μL RB in the chamber and diluting each substrate to 9X, 10X, 11X, and 12X with every injection through ports A to D, respectively.

Once loaded, the sensor cartridge was placed into the Seahorse XFe24 Flux Analyzer for automated calibration. Seahorse Standard XFe24 assay plates were used for loading mitochondria. Initially, total mitochondria were diluted to 10 μg/50 μL in RB, and 50 μL was loaded in each well resulting in 10 μg mitochondria/well. The assay plates were centrifuged at 3,000 rpm for 4 min at 4°C to adhere the mitochondria at the bottom of the wells. After centrifugation, 475 μL RB (pre-incubated to 37°C) was added without disturbing the mitochondrial layer to obtain a final volume of 525 μL per well. After the instrument calibration with the sensor cartridge was complete, the utility plate was replaced by the plate loaded with mitochondria for bioenergetics analysis.

The assays were performed under a previously optimized protocol.⁴⁸ Briefly, it involved cyclic steps of mixing, sequential injections of substrates/inhibitors via ports A through D, mixing, equilibration, and measurement of the OCR through fluorimetric optical probes. The data output gives State III respiration in the presence of pyruvate and malate (PM) and ADP (port A) followed by State IV rate in the presence of oligomycin A (port B). Sequentially, it gives uncoupled respiration State (VPM) and State V succinate (Suc) (State V2) in the presence of FCCP (port C) and rotenone plus succinate (port D), respectively. Raw OCR values were used for analysis within a given experiment and reported in all figures. Plate-to-plate and day-to-day variation may result in different absolute OCR values between experiments.