To assess mitochondrial OXPHOS, respiration capacities of isolated mitochondria were measured with a high-resolution respirometer (Oxygraph-2k, Oroboros) (7 (link)). Isolated mitochondria (∼30–100 µg) were placed into the respirometer chamber, filled with 2 mL of MiR05 medium (110 mmol/L sucrose, 60 mmol/L K-lactobionate, 0.5 mmol/L EGTA, 0.1% BSA, 3 mmol/L MgCl2, 20 mmol/L taurine, 10 mmol/L KH2PO4, 20 mmol/L Hepes, pH 7.1), and then incubated with chemicals at 37 °C in the following order (final concentrations are indicated): 1) 2 mM malate, 5 mM pyruvate, 10 mM glutamate (complex I-linked substrates), 2) 10 mM ADP, and 3) 10 mM succinate (complex II-linked substrate) and 0.5 µM rotenone (a complex I inhibitor). The O2 consumption rates (i.e., respiration rates) were expressed as O2 flux normalized to the mitochondrial protein concentration (µg/µL). DatLab software (Oroboros) was used for data acquisition and analysis.
Datlab software
Manufactured by Oroboros
Sourced in Austria
DatLab software is a data analysis tool designed for use with Oroboros respirometers. The software provides a platform for recording, analyzing, and visualizing data from respirometric measurements.
Automatically generated - may contain errors
Lab products found in correlation
Oxygraph 2k, by Oroboros (29 mentions)
O2k oxygraph, by Oroboros (6 mentions)
O2k respirometer, by Oroboros (4 mentions)
Oligomycin, by Merck Group (3 mentions)
Oxygraph 2k chamber, by Oroboros (3 mentions)
Oxygraph 2k high resolution respirometer, by Oroboros (2 mentions)
Oxygraph 2k oxygen electrode, by Oroboros (2 mentions)
Oxygraph, by Oroboros (2 mentions)
Antimycin a, by Merck Group (2 mentions)
Oxygraph 2k instrument, by Oroboros (1 mentions)
2 nbdg, by Thermo Fisher Scientific (1 mentions)
L lactate assay kit, by Cayman Chemical (1 mentions)
Two chamber oxygraph, by Oroboros (1 mentions)
O2k fluo led2 module, by Oroboros (1 mentions)
Prism 5, by GraphPad (1 mentions)
Prism software, by GraphPad (1 mentions)
Oxygraph 2k respirometer, by Oroboros (1 mentions)
Saponin, by Merck Group (1 mentions)
Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone, by Merck Group (1 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (1 mentions)
85 protocols using datlab software
1
Measuring Mitochondrial Respiration Capacity
2
Mitochondrial Respiration in Drosophila
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Mitochondrial respiration was assayed at 30 °C by high-resolution respirometry using a Oxygraph-2k high-resolution respirometer (OROBOROS Instruments) using a chamber volume set to 2 mL. Calibration with the air-saturated medium was performed daily. Data acquisition and analysis were carried out using Datlab software (OROBOROS Instruments). Five flies per genotype were homogenised in Respiration Buffer [120 mM sucrose, 50 mM KCl, 20 mM Tris–HCl, 4 mM KH2PO4, 2 mM MgCl2, and 1 mM EGTA, 1 g L−1 fatty acid-free BSA, pH 7.2]. For coupled (state 3) assays, complex I-linked respiration was measured at saturating concentrations of malate (2 mM), glutamate (10 mM) and adenosine diphosphate (ADP, 2.5 mM). Complex II-linked respiration was assayed in Respiration Buffer supplemented with 0.15 µM rotenone, 10 mM succinate and 2.5 mM ADP. The addition of proline to the respiration buffer can increase respiration rate in insect samples but was not included here. Respiration was expressed as oxygen consumed per fly. Flies’ weight was equal in all genotypes tested.
3
Mitochondrial Respiration Assay
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The mitochondrial respiratory function was assessed using high-resolution respirometry (Oroboros Oxygraph-2k Ltd.–Oroboros Instruments, Innsbruck, Austria) at 37 °C. Mitochondrial respiratory rates were attained by following a substrate-uncoupler-inhibitor titration (SUIT) protocol, which was adapted in order to obtain both the separate and conjunctive electron flow from complex I (CI) or/and complex II (CII). For the analysis, 1 × 106/mL cells were suspended in mitochondrial respiration medium (MiR05: 110 mM sucrose, 3 mM MgCl2.6H2O, 0.5 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 1 g/L Bovine Serum Albumin (BSA), adjusted to pH 7.1 at 37 °C) [41 (link)]. Data acquisition and analysis was performed with DatLab software (Oroboros Instruments, Innsbruck, Austria). The specific oxygen flux was expressed as pmol/s per cm3 volume.
4
Mitochondrial Respiratory Function Assay
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Myocardial (left ventricle) or skeletal muscles were dissected according to Reference (35 (link)). Mitochondrial respiratory function was measured in saponin (50 µg/ml)-permeabilized muscle fibers by high-resolution respirometry at 30°C, using two-channel titration-injection respirometers (Oroboros Oxygraph) and expressed in pmols oxygen per s, per milligram wet weight. The respiration medium consisted of 110 mm sucrose, 60 mm K-lactobionate, 0.5 mm EGTA, 1 g/l BSA (essentially fatty acid free), 3 mm MgCl2, 20 mm taurine, 10 mm KH2PO4 and 20 mm HEPES, pH 7.1. DatLab software (Oroboros Instruments) was used for data acquisition and analysis. Respiration was stimulated by 1 mm ADP (maximum rate, State 3 respiration) and measured with 10 mm glutamate and 5 mm malate (substrates for Complex I of the mitochondrial respiratory chain). Apparent Km for ADP was measured by ADP titration (step-wise increase in ADP concentration) in the range of 0–2 mm ADP. Km calculation was performed by hyperbolic fit using ‘Sigma Plot’ software.
5
Respiratory System Enzyme Activity Analysis
6
Bioenergetic Assessment of Skeletal Muscle
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After excision, extensor digitorum longus (EDL) muscles were immersed in cold BIOPS (10 mmol/L EGTA, 50 mmol/L 2-[N-morpholino]ethanesulfonic acid, 0.5 mmol/L dithiothreitol, 6.56 mmol/L MgCL2, 5.77 mmol/L ATP, 20 mmol/L imidazole, and 15 mmol/L phosphocreatine [pH 7.1]), and the fibers were separated on ice using two forceps and a dissection microscope. Fibers were permeabilized at 4°C for 30 min using BIOPS solution containing 50 µg/mL saponin. Then the fibers were washed for 10 min in ice-cold mitochondrial respiration solution (MIR05; 0.5 mmol/L EGTA, 3 mmol/L MgCl2, 60 mmol/L K-lactobionate, 20 mmol/L taurine, 10 mmol/L KH2PO4, 20 mmol/L HEPES, 110 mmol/L sucrose, and 1 g/L BSA [pH 7.1]), blotted dry, weighed (3–5 mg), and placed in the Oxygraph-2k chamber (OROBOROS Instruments, Innsbruck, Austria) containing 2 mL of 37°C MIR05. To measure O2 flux, the following substrates were added sequentially: 5 mmol/L malate, 10 mmol/L glutamate, 2.5 mmol/L ADP, 10 mmol/L succinate, three pulses of 0.5 μmol/L carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), 0.5 μmol/L rotenone, and 2.5 μmol/L actinomycin A. A period of stabilization followed the addition of each substrate, and oxygen flux per mass was recorded using DatLab software (OROBOROS Instruments).
7
Mitochondrial Oxygen Consumption Analysis
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Oxygen consumption was performed polarographically using an Oroboros O2k simultaneous to determination of ΔΨm, thus conditions were identical. The oxygen concentration (µM) and oxygen flux (pmol·s–1·mg–1; negative time derivative of oxygen concentration, divided by mitochondrial mass per volume and corrected for instrumental background oxygen flux arising from oxygen consumption of the oxygen sensor and back-diffusion into the chamber) were recorded using DatLab software (Versions 7.3.0.3 Oroboros Instruments, Innsbruck, Austria).
8
Oxygen Consumption Profiling of Cells
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O2 consumption rate (OCR) was determined by high-resolution respirometry using an Oroboros Oxygraph-2 k instrument (OROBOROS® INSTRUMENTS GmbH, Innsbruck, Austria). Cells (control or ISL-treated) were seeded at 4 × 106 cells. The cells were centrifuged at 1000 rpm for 4 minutes and resuspended in MIR05 buffer (Oroboros lab). The respiration experiments were conducted at 37°C in MIR05 buffer. A standard protocol using malate (2 mM), glutamate (10 mM), oligomycin (2 μg/ml), FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) (0.45 μM), succinate (10 mM), digitonin (3.68 μM), rotenone (0.5 μM) and antimycin A (2.5 μM) was used for each measurement. Cellular O2 was calculated from the recorded data as the time derivative of the oxygen content in the chamber; O2 concentrations were calculated using DatLab software (Oroboros Instruments).
9
Mitochondrial Respiration and Metabolism Assays
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Mitochondrial fractions were prepared from cultured cells as we previously described [72 (link)]. Mitochondrial respiration assays using freshly isolated mitochondria were performed by measuring O2 consumption in a 2-channel respirometer (Oxygraph-2k with DatLab software; Oroboros Instruments, Innsbruck, Austria) as we previously described [10 (link), 11 (link), 15 (link), 72 (link)]. Glucose uptake experiments were carried out as previously described [10 (link), 11 (link)] using 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Life Technologies, Grand Island, NY; catalog # N13195). Lactate accumulation in the culture medium was determined using a fluorescence-based L-lactate assay kit from Cayman (Ann Arbor, MI) according to the manufacturer's instructions. Mitochondrial complex activities were determined, with results normalized to citrate synthase activity, as previously described [10 (link), 55 (link), 73 (link)].
10
Measurement of Cardiac Mitochondrial Function
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All the procedures for measurement of freshly cardiac mitochondrial function in vivo were as stated by ours and others’ publications [61 (link),62 (link),63 (link)]. Briefly, ~10 mg heart pieces were immersed in pre-cold BIOPS buffer. Permeabilization of the cardiomyocyte membrane was done via transferring cardiac myofiber to 2 mL of MIR05 buffer containing saponin (50 μg/mL) for 30 min at 4 °C. About 2 mg of permeabilized cardiac myofiber were used for measuring mitochondrial respiration function with a pre-calibrated air saturated MIR05 buffer using Oroboros oxygraph-2k (O2K) system (Oroboros, Innsbruck, Austria) by the consecutive addition of substrates and uncouplers. The leak respiration (state 2) was achieved by the addition of 1.5 mM octanoyl-l-carnitine, 5 mM pyruvate, 10 mM glutamate, and 2 mM malate (P+G+M) into the O2K chamber; state 3 respiration driven by mitochondrial CI substrates was achieved by the titration of 5 mM ADP; and state 3 respiration driven by mitochondrial complex II (CII) substrate was achieved by the addition of 10 mM succinate. To test the competence of the mitochondrial outer membrane, 10 μM cytochrome c was added. Finally, uncoupled OXPHOS was achieved by the addition of 5 μM of carbonyl cyanide m-chlorophenylhydrazone (CCCP). All data were analyzed by Oroboros DatLab software.
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