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O2k respirometer

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The O2k-Respirometer is a modular system for high-resolution respirometry. It measures oxygen consumption and carbon dioxide production in biological samples, providing quantitative data on mitochondrial and cellular respiration.

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Lab products found in correlation

Datlab software, by Oroboros (4 mentions) Pierce quantitative peroxide assay kit, by Thermo Fisher Scientific (1 mentions) Mir05 kit, by Oroboros (1 mentions)

16 protocols using o2k respirometer

1

Mitochondrial Respiration in Adipose Tissue

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Mitochondrial respirometry rates were determined in fresh permeabilized adipose tissue (iWAT) samples using O2K respirometer (Oroboros Instruments). In brief, the finely minced fat depot was placed in ice-cold BIOPS buffer (10 mM Ca-EGTA buffer, 0.1 μM free calcium, 5.77 mM ATP, 6.56 mM MgCl2, 20 mM taurine, 15 mM phosphocreatine, 20 mM imidazole, 0.5 mM DTT, and 50 mM MES hydrate). The tissue was permeabilized with Saponin (final concentration 100μg/ml) and then incubated on a rotational mixer for 30 mins at 4°C. The fat tissue was washed several times with Saponin through 3 cycles of centrifugation (5000xg, 4C, 5 minutes) and replaced with fresh BIOPS buffer for 5 minutes on the rotational mixer. Finally, the fat depot was transferred to Oroboros O2K Respirometer, which contained 2ml Mir05 respiration medium and the chamber was sealed. Once baseline respiration was achieved, the tissue was subjected to titrations of substrates (10μM cytochrome C, 5mM pyruvate, 2mM malate, 10mM glutamate, 2mM ADP, 2.5μM oligomyin, 1.5μM carbonyl cyanide-4-phenylhydrazone (FCCP) in 0.5μM increments, and 2.5μM antimycin A). Oxygen consumption was normalized to tissue weight.
Mooli R.G., Mukhi D., Watt M., Edmunds L., Xie B., Capooci J., Reslink M., Eze C., Mills A., Stolz D.B., Jurczak M, & Ramakrishnan S.K. (2020). Sustained mitochondrial biogenesis is essential to maintain caloric restriction-induced beige adipocytes. Metabolism: clinical and experimental, 107, 154225.
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2

Mitochondrial Respiration in Adipose Tissue

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Mitochondrial respiratory rates were determined in fresh permeabilized adipose tissue samples using an O2K respirometer (Oroboros Instruments, Innsbruck, Austria). All measurements were performed in a respiration buffer at 37°C. O 2 concentration was maintained between 200-400 μM in the respiration buffer, which was stirred rigorously by a magnetic stir bar to minimize any O2 dependency artifacts from the data. Titrations of substrates (pyruvate, octanoyl-l-carnitine, glutamate, malate and succinate) followed by the UCP1 inhibitor GDP were used to directly assay UCP1 function. Further details on respirometric analysis and the respiration protocols used can be found in the Supplemental Information.
Porter C., Herndon D.N., Chondronikola M., Chao T., Annamalai P., Bhattarai N., Saraf M., Capek K., Reidy P.T., Daquinag A.C., Kolonin M.G., Rasmussen B.B., Borsheim E., Toliver-Kinsky T, & Sidossis L.S. (2016). Human and mouse brown adipose tissue mitochondria have comparable UCP1 function. Cell metabolism, 24(2), 246-255.
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3

Evaluating Platelet Mitochondrial Bioenergetics

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Mitochondrial bioenergetics in platelets was evaluated by the high-resolution respirometry (HRR) method with the use of an O2k-Respirometer (Oroboros Instruments, Innsbruck, Austria) [38 (link),39 (link)]. For the evaluation of platelet mitochondrial bioenergetics, substrate-uncoupler-inhibitor (SUIT) protocol 1 was applied [40 (link)] as described in detail previously [12 (link)].
Gvozdjáková A., Kucharská J., Rausová Z., Lopéz-Lluch G., Navas P., Palacka P., Bartolčičová B, & Sumbalová Z. (2023). Effect of Vaccination on Platelet Mitochondrial Bioenergy Function of Patients with Post-Acute COVID-19. Viruses, 15(5), 1085.
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4

Platelet Mitochondria Respiration Measurement

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Platelet mitochondria respiration (pmol O2 s-1·10−6 platelets) was measured using an O2k-respirometer (Oroboros Instruments, Innsbruck, Austria). Background calibration was performed prior to each experiment as described elsewhere (Rose et al., 2019 (link)). To allow the exchange of substrates between the cytosol and respiration medium, PL plasma membranes were selectively permeabilized with digitonin (Gnaiger, 2020 (link)). PL mitochondria respiration was evaluated using a substrate-uncoupler-inhibitor titration (SUIT) reference protocol (Table 1).
Diaz E.C., Adams S.H., Weber J.L., Cotter M, & Børsheim E. (2023). Elevated LDL-C, high blood pressure, and low peak O2 associate with platelet mitochondria function in children—The Arkansas Active Kids Study. Frontiers in Molecular Biosciences, 10, 1136975.
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5

Mitochondrial Respiration in Platelets

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Mitochondrial respiration was measured with high-resolution respirometry method [46 (link),47 ]. For respirometric analysis, 200 × 106 platelets were used in a 2 mL chamber of an O2k-respirometer (Oroboros Instruments, Austria). The respiration was measured in mitochondrial respiration medium MiR05 [48 (link)] with 20 mM creatine at 37 °C under continuous stirring at 750 rpm. Two different substrate–uncoupler–inhibitor titration (SUIT) protocols were applied [17 ,18 ,49 ] with common cross-linked respiratory states, allowing for harmonization of both protocols. The data were collected with DatLab software (Oroboros Instruments) using a data recording interval of 2 s [48 (link)]. All substrates and inhibitors were added in saturating concentrations, and the uncoupler was titrated in optimum concentration to reach the maximum O2 flow at given respiratory state. The O2 flow after Gp addition is 20–30% lower than the respiratory capacity, as additional uncoupler titration is necessary to reach the maximum O2 flow at this state.
Palacka P., Gvozdjáková A., Rausová Z., Kucharská J., Slopovský J., Obertová J., Furka D., Furka S., Singh K.K, & Sumbalová Z. (2021). Platelet Mitochondrial Bioenergetics Reprogramming in Patients with Urothelial Carcinoma. International Journal of Molecular Sciences, 23(1), 388.
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6

Monitoring Sulfide Oxidation by SQOR

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Human SQOR was purified and embedded in nanodiscs as described previously (27 (link)). O2 consumption by FADH2 in ndSQOR was monitored using an O2k respirometer (Oroboros Instruments), equipped with two polarographic O2-sensing electrodes housed in separate 2 ml chambers. Each chamber was filled with 100 mM potassium phosphate, pH 7.4, and sulfide (100 μM) and sulfite (200 μM) were added before sealing the chambers and pre-incubating for ∼5 min at 25 °C. The reaction was initiated by injecting ndSQOR (100 nM) and monitored over a period of ∼10 min. Initial O2 concentrations were varied by aerating N2-purged buffer in the chambers before sealing when the desired O2 concentration was reached. H2O2 production was assayed using the Pierce Quantitative Peroxide Assay Kit (Thermo Fisher) according to the manufacturer’s protocol.
Kumar R., Landry A.P., Guha A., Vitvitsky V., Lee H.J., Seike K., Reddy P., Lyssiotis C.A, & Banerjee R. (2021). A redox cycle with complex II prioritizes sulfide quinone oxidoreductase-dependent H2S oxidation. The Journal of Biological Chemistry, 298(1), 101435.
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7

Measuring UCP1-Dependent Respiration in BAT

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BAT mitochondria were assessed for UCP1-dependent respiration in freshly isolated samples using the O2k respirometer (Oroboros Instruments, Innsbruck, Austria). All assays were conducted in the K-medium (10 mM Tris, 80 mM KCl, 3 mM MgCl2, 5 mM KH2PO4, 0.5 mM EDTA) at 25 °C. UCP1-dependent respiration was directly evaluated by titration of substrates (5 mM pyruvate, 1.5 mM octanoyl-l-carnitine, 10 mM glutamate, 2 mM malate, 10 mM succinate) followed by titration of the UCP1 inhibitor ADP (10–15 mM) and the ATP synthase inhibitor oligomycin (2 µg/ml)67 (link). No data were excluded from the analyses.
Marvanova A., Kasik P., Elsnicova B., Tibenska V., Galatik F., Hornikova D., Zvolska V., Vebr P., Vodicka P., Hejnova L., Matous P., Szeiff Bacova B., Sykora M., Novotny J., Neuzil J., Kolar F., Novakova O, & Zurmanova J.M. (2023). Continuous short-term acclimation to moderate cold elicits cardioprotection in rats, and alters β-adrenergic signaling and immune status. Scientific Reports, 13, 18287.
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8

Substrate-Specific Mitochondrial Respiration

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Different substrate oxidation effects on respiration were evaluated using inhibitors for the three primary substrates that fuel mitochondria: glucose/pyruvate (12 μM UK5099, an inhibitor of the mitochondrial pyruvate carrier, MPC), glutamine (8 μM BPTES, an inhibitor of glutaminase 1, GLS-1), and long-chain fatty acids (14 μM etomoxir, an inhibitor of carnitine palmitoyl transferase 1aCPT1a). Oxygen consumption was measured in AML-12 cells in suspension using a high-resolution O2k respirometer (Oroboros Instruments). About 1 × 106 cells were incubated in 2 ml of the same medium used in Seahorse experiments in the absence or presence of 2 μM TG and/or inhibitors, at 37 °C with continuous stirring (400 rpm). After basal respiration, 1 μM oligomycin was injected, followed by 5 μM CCCP, to calculate maximal respiration.
Vilas-Boas E.A., Cabral-Costa J.V., Ramos V.M., Caldeira da Silva C.C, & Kowaltowski A.J. (2023). Goldilocks calcium concentrations and the regulation of oxidative phosphorylation: Too much, too little, or just right. The Journal of Biological Chemistry, 299(3), 102904.
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9

Mitochondrial Function in Mouse Hearts

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Mitochondrial function in manually dissected fibers from the left ventricular free wall of mouse hearts were evaluated using an Oroboros O2k-Respirometer. Heart samples weighed between 0.5 and 1.0 mg. Serum (5%) from the control and helium-exposed animals was incubated in the oximetry chamber along with ventricular tissue in media containing: 100 mM KCl, 75 mM mannitol, 25 mM sucrose, 5 mM H3PO4, 0.05 mM EDTA and 10 mM Tris HCl; pH 7.2, at 37 °C. After 10 min of equilibration, respiratory function of complex I and II activity (CI, CII), maximum oxidative phosphorylation capacity (mOX) and maximum uncoupled capacity (mUC) were determined after addition of complex I substrates, glutamate and malate. Oxygen use trace and rate determinations were obtained using DatLab 7 software and normalized to protein.
Weber N.C., Schilling J.M., Warmbrunn M.V., Dhanani M., Kerindongo R., Siamwala J., Song Y., Zemljic-Harpf A.E., Fannon M.J., Hollmann M.W., Preckel B., Roth D.M, & Patel H.H. (2019). Helium-Induced Changes in Circulating Caveolin in Mice Suggest a Novel Mechanism of Cardiac Protection. International Journal of Molecular Sciences, 20(11), 2640.
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10

Mitochondrial Respiration Profiling in Platelets

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Oxygen consumption in the intact and permeabilized platelets and the capacity of oxidative phosphorylation (OXPHOS) at Complex I were determined with the HRR method. HRR is a sensitive technique to determine mitochondrial bioenergetic function in human platelets that are isolated from peripheral blood [20 (link),21 (link)]. For mitochondrial respirometric analysis, 200 × 106 platelets were used in a 2 mL chamber of an O2k-Respirometer (Oroboros Instruments, Innsbruck, Austria). The respiration was measured at 37 °C in a mitochondrial respiration medium, MiR05 [20 (link)] and 20 mM creatine under continuous stirring at 750 rpm. The data were collected with the DatLab software (Oroboros Instruments, Innsbruck, Austria) with a data recording interval of 2 s.
Gvozdjáková A., Sumbalová Z., Kucharská J., Komlósi M., Rausová Z., Vančová O., Számošová M, & Mojto V. (2020). Platelet Mitochondrial Respiration, Endogenous Coenzyme Q10 and Oxidative Stress in Patients with Chronic Kidney Disease. Diagnostics, 10(3), 176.
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