Xfe96 flux analyzer

OCR and ECAR were measured using the XF^e96 flux analyzer (Seahorse Bioscience, Agilent Technologies LDA UK Ltd, Stockport, UK) and the XF Cell Mito Stress Test kit (Seahorse Bioscience, Agilent Technologies LDA UK Ltd), according to the manufacturer's instructions. In all, 8x10⁴ cells were seeded onto poly-d-lysine-coated XF microplates. Briefly, metabolic flux measurements were assessed under basal conditions and in response to the ATP synthase inhibitor, oligomycin (2.5 μM), the electron transport chain (ETC) accelerator, FCCP (1 μM), and finally the ETC complex 1 and 3 inhibitors, antimycin A and rotenone (2.5 μM), respectively. Data were analyzed using the XF software (Seahorse Bioscience, Agilent Technologies LDA UK Ltd).

Cytochrome c is an important protein of the respiratory chain and loss can lead to reduction in respiratory activity. Exogenous cytochrome c addition may rescue mitochondrial function (23 (link), 24 (link)). Therefore, permeabilized HL-1 cardiomyocytes were measured via Seahorse XFe96 Flux Analyzer after pre-incubation with IPA for 24 h as previously described. 1 mM exogenous cytochrome c (cytochrome c from equine heart; final concentration: 100 μM) was simultaneously injected with saponin (final concentration of 250 μg/ml), 4 mM ADP, 10 mM succinate, and 2 μM rotenone. After two baseline measurements, respiratory activity was measured five times with cycles of mix (0.5 min)/wait (0.5 min)/measure (2 min). At the end of the measurement a mix of 20 μM rotenone/antimycin A (final concentration: 2 μM) was injected to the cells.

Forty-eight hours after transfection, oxygen consumption rates were measured using a Seahorse XFe96 Flux Analyzer® following the manufacturer’s protocol. Oligomycin was injected into port A to a final concentration of 2 μM, FCCP was injected into port B to a final concentration of 6 μM, and Rotenone/Antimycin A (RAA) was injected in Port C to a final concentration of 4 μM rotenone/4 μM Antimycin A. Maximal respiration was calculated by subtracting non-mitochondrial respiration from the rate after injection of FCCP. After measurement of oxygen consumption rates, cells were lysed in 30 μl 1X SDS loading buffer. The lysate was resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were probed with HRP-conjugated anti-β-Actin (Santa Cruz Biotechnology). Band density was quantified using Image J. OCR data was normalized by the density of β-Actin. N is expressed as the number of wells per treatment group.

To determine whether pyruvate was used as an oxidizable substrate in WT and Pparα^-/- retinas, the retinal OCR was measured after addition of pyruvate using a Seahorse XFe96 Flux Analyzer®. Mice were euthanized with ketamine/xylazine, whole retinas were isolated and 1 mm punch biopsies were loaded into a 96-well spheroid plate. Retinal punches were incubated in assay media consisting of DMEM 5030 supplemented with 5 mM glucose, 0.5 mM pyruvate, 2 mM glutamine, and 10 mM HEPES. Assay media (vehicle) control or high-pyruvate-containing assay media were injected into port A to a final concentration of 5 mM pyruvate in pyruvate-injected punches, and 0.5 mM pyruvate in vehicle-injected punches. Oligomycin was injected into port B to a final concentration of 1 μM, FCCP was injected into port C to a final concentration of 2 μM, and RAA was injected in Port D to a final concentration of 2 μM rotenone/2 μM antimycin A. Maximal respiration was calculated by subtracting non-mitochondrial respiration from the rate after injection of FCCP. Fold change in maximal respiratory rate was calculated by dividing the maximal rate of pyruvate-injected punches by that of vehicle-injected punches for both genotypes. N is expressed as the number of retinal punches per treatment group.

A549 cells were treated in triplicate with serially diluted IM for 24 h and were washed prior to the OCR measurements. In the reprogramming experiments, OSKM-transduced MEFs were reseeded in triplicate at a density of 3 × 10³ cells per well in poly-l-lysine (Sigma)-coated 96-well XF plates (Agilent, Santa Clara, California, USA) 4 days after reprogramming. On the following day (day 5), the medium was replaced with mESC medium with or without the test chemicals. On day 7, OCR/ECAR was measured using a Seahorse XFe96 Flux analyzer according to the manufacturer’s instructions. The probe cartridge was calibrated without CO₂ for 1 h, and then basal OCR/ECAR measurement was performed. The following ETC-targeting compounds were sequentially added at each indicated time point: 1.5 μM oligomycin (ATP synthase, complex V, inhibitor), 5 μM FCCP (uncoupler), and 0.5 μM rotenone (complex I inhibitor) + antimycin A (complex III inhibitor). The value was normalized against the cell number.

An XFe96 flux analyzer (Seahorse Biosciences, Agilent) was used to analyze the mitochondrial OCR. Briefly, 5 × 10³ cells were plated into XF‐96 cell culture microplates in 80 μL of Dulbecco's modified Eagle medium and incubated at 37°C overnight. Cell mitochondria stress test was conducted by adding oligomycin (1.5 μM), trifluoromethoxy carbonylcyanide phenylhydrazone (1.0 μM), and rotenone/antimycin A (both 0.5 μM). Hoechst was used for nuclear staining. OCR values were normalized by cell counting (Cytation 7, Cell Imaging Multi‐Mode Reader, Agilent BioTek).

To determine whether mitochondrial respiration was changed in WT and Pparα^-/- retinas after onset of neurodegeneration, retinal oxygen consumption rate (OCR) was measured in 8- and 12-week-old retinas using a Seahorse XFe96 Flux Analyzer®. Mice were euthanized with ketamine/xylazine, whole retinas were isolated and 1 mm punch biopsies were loaded into a 96-well spheroid plate. Retinal punches were incubated in assay media consisting of DMEM 5030 supplemented with 12 mM glucose, 5 mM pyruvate, 2 mM glutamine and 10 mM HEPES. Oligomycin was injected into port A to a final concentration of 2 μM, FCCP was injected into port B and C to a final concentration of 1 μM, and Rotenone/Antimycin A (RAA) was injected in Port D to a final concentration of 2 μM rotenone/2 μM Antimycin A. Basal respiration was calculated by subtracting non-mitochondrial respiration from the basal rate prior to injection of Oligomycin. Maximal respiration was calculated by subtracting non-mitochondrial respiration from the rate after injection of FCCP. N is expressed as the number of retinal punches per treatment group.