The OCR of permeabilized neurons and isolated brain mitochondria (from 9-month-old mice) was measured by the XF96 Extracellular Flux analyzer (Seahorse Bioscience). For assays using cultured neurons, primary neurons were dissociated from the forebrains of P0 pups, seeded at 1.9 × 104 per well (PDL-coated XF96 plate), cultured to DIV13, and incubated with dyclonine or DMSO (0.1%) for 24 hours. Before the start of the OCR measurement, all but 30 μl of Neurobasal-A/B27 culture medium was removed from each well. Cells were washed twice with prewarmed MAS-BSA assay medium [fatty acid–free BSA (4 mg/ml), pH 7.2] and incubated in 180 μl at 37°C for 5 min. Following the cartridge calibration, cells were loaded into the XF96 Extracellular Flux analyzer and further equilibrated for 10 min with two cycles of 3-min mixing and 2-min resting before the first measurement of basal respiration. OCR was measured at 37°C under basal conditions followed by the sequential injection of prewarmed 10× mitochondrial substrates/ADP (20 μl), oligomycin (22 μl), FCCP (24 μl), and rotenone/antimycin A (26 μl). The final concentrations of injected compounds were as follows: 10 mM mitochondrial substrates (pyruvate/malate or succinate), 1 mM ADP, 1.5 μM oligomycin, 3 μM FCCP, and 2 μM rotenone/antimycin A. Saponin (25 μg/ml) was coinjected with substrate/ADP to permeabilize the cells and stimulate ADP-dependent respiration. Two baseline measurements were obtained before any injection, and two response measurements were collected after each injection except for after FCCP addition, which consisted of one measurement (nine total measurements in each assay). Each measurement cycle consisted of 2-min mixing, 2-min waiting, and 3-min data acquisition. This protocol allowed for sequential assessment of basal cell respiration, maximal mitochondrial respiratory capacity (State 3 with substrate/ADP), proton leak (State 4o with oligomycin), uncoupled maximal respiration (State 3U with FCCP), and nonmitochondrial respiration with the complex I/complex III inhibitors rotenone/antimycin A. Cells were washed twice with MAS buffer to remove residue BSA contained in the assay medium and lysed in 50 μl of lysis buffer [25 mM Hepes, 1 mM EGTA, 1 mM EDTA, 0.1% SDS, 1% NP-40, 1× protease, and phosphatase inhibitor (pH 7.0 adjusted with NaOH)], and total protein was determined by BCA assay. Oxygen consumption was normalized to total protein content as pmol O2 min−1 μg−1 total protein.

For OCR measurements of isolated brain mitochondria, the mitochondria were diluted to 3 μg/20 μl in cold MAS-BSA buffer + substrate (pyruvate/malate or succinate, to maintain healthy state of mitochondria) and plated onto 96-well PDL-coated plates (20 μl per well). Wells without mitochondria were used for background correction. The plate was centrifuged at 2000g for 20 min at 4°C to attach the mitochondria. After centrifugation, an additional 160 μl of MAS-BSA buffer + substrate was added to the wells. Mitochondria were checked under the microscope to ensure a homogeneous mitochondrial monolayer in each well and incubated at 37°C for 10 min. The plates were further equilibrated for 8 min by two cycles of 1-min mixing and 3-min rest before the measurement of basal respiration. Two baseline measurements were obtained before any injection, and one response measurement was obtained after each injection followed by additional 30-s mixing. The final concentrations of compounds after injections were as follows: 10 mM pyruvate/malate or succinate, 1 mM ADP, 2 μM oligomycin, 4 μM FCCP, and 2 μM rotenone/antimycin A. Each measurement cycle consisted of 30-s mixing and 3-min data acquisition except for the measurement after ADP injection, which lasted 6 min to observe the transition from State 3 to State 4 due to the depletion of ADP in the microchamber. The additional mixing step after each measurement facilitated the sensor returning to ambient O2 concentration. State 3 respiration parameters driven by mitochondrial complex I substrates (pyruvate/malate) were measured first, while complex II–driven respiration (succinate) was measured by inhibiting complex I with rotenone (2 μM). OCR was calculated by the Seahorse XF96 software package. OCR data measured from isolated brain mitochondria are displayed in the “point-to-point mode” showing a series of OCRs across each measurement period.

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