Populations of 1,000 adult nematodes were thoroughly washed, immersed in M9 buffer into the chamber of an Oroboros O2k Oxygraph (O2k Oxygraph, Oroboros Instruments, Innsbruck, Austria), and oxygen flux measured at 20°C. The provided DatLab software (Version 7.0.0.2, Oroboros Instruments, Innsbruck, Austria) was used for analysis.
O2k oxygraph
Manufactured by Oroboros
Sourced in Austria
The O2K Oxygraph is a high-resolution respirometry system designed for the analysis of cellular respiration. It measures oxygen consumption and carbon dioxide production in tissue samples, cells, and isolated mitochondria. The instrument provides accurate and reliable data on respiratory function.
Automatically generated - may contain errors
Lab products found in correlation
Datlab software, by Oroboros (6 mentions)
Amplex ultrared 10 μm horseradish peroxidase 3 u ml detection system, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Datlab 4, by Oroboros (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Datalab software, by Oroboros (1 mentions)
Mitokit cii, by Oroboros (1 mentions)
Rf 5301pc, by Shimadzu (1 mentions)
Amplex ultrared, by Thermo Fisher Scientific (1 mentions)
Mir05 medium, by Oroboros (1 mentions)
Ab3242, by Merck Group (1 mentions)
Cell signaling 4259, by Cell Signaling Technology (1 mentions)
Datlab 7, by Oroboros (1 mentions)
Clark type electrode, by Hansatech (1 mentions)
Mir05, by Oroboros (1 mentions)
Datlab6, by Oroboros (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Percoll, by Thermo Fisher Scientific (1 mentions)
Oligomycin, by Merck Group (1 mentions)
90 protocols using o2k oxygraph
1
Nematode Oxygen Flux Measurement
2
Saponin-Permeabilized Muscle Fiber Respiration
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At the end of incubation time, fibers (10 mg) were transferred into 2mL of fresh ice-cold BIOPS relaxing solution and 20μL of saponin (freshly-prepared stock solution 5mg/mL) are added to obtain final concentration of 50μM. Bundles were maintained for 30 min with saponin solution, and then washed for 5 min three times in ice-cold respiration medium MiRO5 containing 110 mM sucrose, 20 mM HEPES, 10 mM KH2PO4, 20 mM taurine, 3 mM MgCl2 6H2O, 60 mM MES-K, 0.5 mM EGTA and 0.1% bovine serum albumin; pH 7.1. Respiration rates were measured (O2k oxygraph, Oroboros, Innsbruck, Austria) and expressed in picomoles O2 per second per milligram wet weight. Data acquisition and analysis were performed with Datlab4 software (Oroboros, Innsbruck, Austria).
Substrate and/or inhibitors for respiratory experiments were added within MiRO5 in a step-by-step manner using micro syringes. Chemical agents (Sigma Aldrich, France) were sequentially prepared according to Oroboros manufacturer data sheet and administered as we have previously described [12 (link)]. Respiration rates were measured (O2k oxygraph, Oroboros, Innsbruck, Austria) and expressed in picomoles O2 per second per milligram wet weight.
Chemical agents (Sigma Aldrich, France) were sequentially prepared according to Oroboros manufacturer data sheet and administered as described below:
Substrate and/or inhibitors for respiratory experiments were added within MiRO5 in a step-by-step manner using micro syringes. Chemical agents (Sigma Aldrich, France) were sequentially prepared according to Oroboros manufacturer data sheet and administered as we have previously described [12 (link)]. Respiration rates were measured (O2k oxygraph, Oroboros, Innsbruck, Austria) and expressed in picomoles O2 per second per milligram wet weight.
Chemical agents (Sigma Aldrich, France) were sequentially prepared according to Oroboros manufacturer data sheet and administered as described below:
3
Measuring Mitochondrial Respiration and H2O2 Production
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Hindlimb muscle mitochondrial respiratory function was tested using the gastrocnemius muscle in the hindlimb and was isolated as previously reported.22 (link),24 Using the CK clamp system described, respiratory function was assessed at 37°C in buffer Z (in mmol/L) supplemented with creatine monohydrate (5 mM), using the O2K Oxygraph (Oroboros Instruments). Isolated mitochondria were energized with pyruvate and malate (5 mM and 2.5 mM, respectively) to obtain state 2 respiration, followed by addition of the CK clamp and the addition of PCr to titrate the energy demand from a mimicked high exercise level to resting levels. The slope of the relationship between the cellular energy demand (ΔGATP) and oxygen consumption (JO2) was calculated. The rate of respiration was expressed as pmol/s/mg of mitochondria. All respiration measurements were conducted at 37°C and a working range [O2] of ∼200 μM. H2O2 production was assessed using pyruvate and malate and identical energy demands as performed using the O2K Oxygraph (Oroboros Instruments) via the Amplex UltraRed (10 μM)/horseradish peroxidase (3 U/mL) detection system (Thermo Fisher Scientific), as previously described.21 (link),24
4
Mitochondrial Respiration Profiling
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High-resolution O2 consumption measurements42 (link) were conducted at 37 °C in buffer Z (in mmol/l) (105 K-MES, 30 KCl, 1 EGTA, 10 K2HPO4, 5 MgCl26H2O, 0.5 mg/ml BSA, pH 7.1), supplemented with creatine monohydrate (20 mM), using the OROBOROS O2K Oxygraph. A substrate inhibitor titration protocol was performed as follows: 2 mmol/l Malate + 10 mmol/l Glutamate (State 2 respiration), followed by the addition of 4 mmol/l ADP to initiate State 3 respiration supported by Complex I substrates, convergent electron flow through complexes I and II was initiated with the addition of 10 mmol/l Succinate, 10 μmol/l Rotenone was subsequently added to inhibit Complex I, followed by 10 μmol/l Cytochrome C to test the integrity of the mitochondrial membrane, Complex IV supported respiration was examined using the electron donor N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) at 0.4 mmol/l in the presence of 2 mmol/l Ascorbate (to limit auto-oxidation of TMPD) and 5 μmol/l of Antimycin A (to prevent reverse electron flow through Complex III). The rate of respiration was expressed as pmol/sec/mg fiber dry weight. All respiration measurements were conducted at 37 °C and a working range [O2] of ~350 to 200 μM.
5
Mitochondrial Function and Inflammation in Sheep
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Mitochondrial function in right ventricle and renal cortex tissue was performed by high resolution respirometry (O2k-Oxygraph; Oroboros Instruments, Innsbruck, Austria); see method in Additional file 1 . Plasma levels of inflammatory cytokines, hyaluronan and cardiac troponin-I were measured by sheep-specific ELISA as described [28 (link), 37 (link)], or pig-hsCTn-I ELISA (Life Diagnostics). Full blood counts were performed on the Mindray BC-5000 Vet analyser, and viscoelastic tests by ROTEM [28 (link)]. Serum biochemistry and urinalysis was performed by QML-Vetnostics.
6
Mitochondrial Respiration Assessment Protocol
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Respiration experiments were conducted at 20°C using a Clark-type electrode (O2k Oxygraph, Oroboros Instruments, Austria). For each measurement, an aliquot of 80 μL in MirO5-resuspended mitochondria, as described preciously, was inserted into 2 mL of air-saturated MirO5-containing electrode chamber. For analysis, the provided DatLab software (Version 7.0.0.2) was used. To determine mitochondrial function, a complex protocol (developed by Prof. Erich Gnaiger, Oroboros, Innsbruck, Austria) was applied, as previously stated [37 (link)].
7
Mitochondrial Respiration Protocols for Cellular Analysis
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Cells and tissue were prepared for mitochondrial respiration as described previously [9 (link)] before being transferred to respirometer chambers using the Oroboros O2K oxygraph (Oroboros, Innsbruck, Austria). Electron flow through complex I was supported by glutamate + malate (10 mM and 2 mM, respectively) to determine leak oxygen consumption (GML). Following stabilization, adenosine diphosphate (ADP) (2.5 mM) was added to determine oxidative phosphorylation capacity (GMD). Succinate was added (GMSD) for complex I + II electron flow into the Q-junction. Lastly, residual oxygen consumption was measured by adding antimycin A (2.5 μM) to block complex III action, effectively stopping any electron flow, which provides a baseline rate of respiration. Respiratory control ratio (RCR) was determined as the ratio of GMP/GML. Following respiration protocol, samples were removed from the chambers and used for further analysis, including protein quantification.
8
Mitochondrial Respiration Analysis
9
Measuring Mitochondrial Respiration in Tissues
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High-resolution O2 consumption was determined at 37 °C in permeabilized muscle fiber bundles, adipose, and liver using the Oroboros O2K Oxygraph with MiR05 respiration buffer (Oroboros Instruments, Innsbruck, Austria), as described previously [18 (link),19 (link),20 (link),21 (link)]. Before the addition of the sample into respiration chambers, a baseline respiration rate was determined. After the addition of the sample, the chambers were briefly hyperoxygenated to ~250 nmol/mL. Following hyperoxygenation, respiration was determined using all or parts of the following substrate–uncoupler–inhibitor–titration protocol: electron flow through complex I was supported using glutamate + malate (10 and 2 mM, respectively) to determine the leak oxygen consumption (GM). Following stabilization, ADP (2.5 mM) was added to determine the oxidative phosphorylation capacity (D). Succinate was added (S) for the complex I + II electron flow into the Q-junction.
10
Mitochondrial Respiratory Activity Assay
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Oxygen consumption and superoxide production were determined using the high resolution O2k oxygraph (Oroboros Instruments). Freshly isolated RHM (1 mg protein) were resuspended in 2 mL of KCL buffer supplemented with 17.6 U SOD, 8.76 U HRP, 12.5 μM Amplex Red and 3 μM BSA and oxygen consumption and superoxide production were induced by simultaneous addition of 10 mM of succinate in each chamber under the constant stirring and constant temperature (T = 37°C). After 1 min, the indicated compounds and control (EtOH) were added and recording of amperometric and fluorescence changes was continued for 25 min. Obtained results of all measurements are presented as means ± s.e.m. of n = 3, repeated on 4 different occasions.
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