Mitochondrial respiration experiments were adapted from Kuznetsov, et al. [34 (link)]; our methodology for this study is described elsewhere [35 ]. Briefly, whole cerebella from treated and untreated FVB and B05 mice were quickly extracted from CO2-asphyxiated wild type and B05 mice (treated and untreated) under IACUC guidelines and prepared in homogenate buffer (0.25 M sucrose, 0.5 mM EDTA, 50 mM Tris-HCl, pH 7.4), and added to the calibrated respirometer set to 30°C (Oxytherm System, Hansatech Instruments, UK). Tissue plasma membranes were permeabilized in the chamber with digitonin (15 μg/mL). Oxygen consumption was recorded in response to the sequential addition of glutamate/malate (Complex I substrate, 10 mM and 5 mM, respectively), ADP (1 mM), rotenone (Complex I inhibitor, 0.5 μM), succinic acid (Complex II substrate, 10 mM), antimycin A (Complex III inhibitor, 5 μM), TMPD/ascorbate (Electron donor, 0.5 mM and 2 mM, respectively) and cytochrome C (10 μM). Percent of total respiration following activator and inhibitor addition was calculated by normalizing the average oxygen consumption values by cerebellar wet weight and comparing it to the corresponding normalized intra-assay response to TMPD/ascorbate. Three biological replicates per genotype and treatment were averaged ± SEM.
Oxytherm system
Manufactured by Hansatech
Sourced in United Kingdom
The Oxytherm System is a lab equipment product designed for the measurement of oxygen consumption or evolution. It provides precise and reliable data on oxygen levels in a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Lactate assay kit, by Merck Group (3 mentions)
S1 mini clark type electrode disc, by Hansatech (2 mentions)
Oxyt1 electrode control unit, by Hansatech (2 mentions)
Glucose uptake colorimetric assay kit, by Merck Group (1 mentions)
Genesys 10, by Thermo Fisher Scientific (1 mentions)
Seahorse xfe96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
35 protocols using oxytherm system
1
Mitochondrial Respiration Profiling in Mouse Cerebella
2
Photosynthesis and Respiration Assay
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Photosynthetic and respiratory activities were measured as evolution and absorption of oxygen, respectively, using an oxygen electrode (Oxytherm System, Hansatech, Norfolk, UK). The liquid culture of Synechocystis cells were grown at 34C or 24C for 1 d and assayed at the same temperature. Photosynthetic activity in the samples was measured at a light intensity of 600 µmole photons m -2 s -1 , which represented saturated light conditions. Sodium bicarbonate (2.5 mM) was added to the cell suspensions as the carbon source. Respiratory activity was measured under dark conditions.
3
Measurement of Oxygen Consumption Rate
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Oxygen consumption rate in a closed environment was measured using the Oxytherm system (Hansatech). Samples were run at a cell concentration of 1×106 cells/ml for 10 minutes at 37°C in the appropriate culture media for each cell line. All samples were run in triplicate. Further detailed methods were described in Cook et al.[4] (link).
4
Measuring Cellular Metabolism Dynamics
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Cells in 10‐cm dishes grew to about 80% confluence and then were trypsinized and resuspended in 3 mL of culture solution. An Oxytherm System (Hansatech) was applied to detect oxygen consumption.
Glucose uptake and lactate production were measured at 48 hours after cells were laid in 6‐well plates or treated, and then collected and investigated culture medium. Lactate production and glucose uptake were analysed using Lactate Assay Kit (Sigma) and Glucose Assay Kit (Shanghai Rongsheng Biotech), respectively, according to product specification.
Glucose uptake and lactate production were measured at 48 hours after cells were laid in 6‐well plates or treated, and then collected and investigated culture medium. Lactate production and glucose uptake were analysed using Lactate Assay Kit (Sigma) and Glucose Assay Kit (Shanghai Rongsheng Biotech), respectively, according to product specification.
5
Perhexiline Alters Glucose Uptake in CLL Cells
6
Perhexiline Alters Glucose Uptake in CLL Cells
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CLL cells were first treated with 5 μM Perhexiline in triplicates for 3 h in glucose-free medium. [3H]2-deoxyglucose (0.4 μCi/ml) was then added and incubated for 60 min. The cells were collected, washed twice with PBS, and suspended in 200 μl of water, then mixed with 1 ml scintillation fluid. Radioactivity was detected by scintillation countering. Cellular oxygen consumption was measured by an Oxytherm system (Hansatech Instrument, Norfolk, UK).
7
Measuring Cellular Respiration Rates
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For measurements of respiration rates, cells were grown to late log phase in YPGal medium and then washed, counted, and resuspended in fresh YPGal medium at 108 cells per milliliter. The rate of oxygen consumption was then measured at 30 °C by using the Oxytherm system (Hansatech). Cyanide-sensitive respiration was calculated after the addition of 1 mM KCN, and the cyanide-insensitive respiration was subtracted from the total respiration.
8
Measuring Cellular Oxygen Consumption
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Oxygen consumption of 5 × 106 human cells was measured 48 h after transfection, following permeabilization with 80 μg/ml digitonin, in a Clark-type electrode (Hansatech Oxytherm system) using respiratory buffer A22 (link) at 37 °C. Complex II-driven respiration was measured in the presence of 10 mM ADP and 10 mM succinate. AOX-driven (antimycin-resistant) respiration was measured after the further addition of (60 ng/ml) antimycin A, with subtraction of any residual oxygen consumption after adding 100 μM n-propyl gallate. Respirometry on S2 cells was as described previously17 (link) and was also conducted on homogenates from 1–4 day-old Drosophila males. Briefly, 25 males were gently homogenized in 0.8 ml ice-cold isolation buffer (250 mM sucrose, 5 mM Tris-HCl, 2 mM EGTA, pH 7.4) and muslin-filtered. Respirometry was performed on 150 μl aliquots of this homogenate, mixed with 500 μl assay buffer (120 mM KCl, 5 mM KH2PO4, 3 mM HEPES-KOH, 1 mM EGTA, 1 mM MgCl2, 0.2% BSA, pH 7.2), substrates (15 mM glycerol-3-phosphate and 5 mM ADP) and inhibitors as for permeabilized mammalian cells.
9
Measuring OXPHOS Complex Inhibition
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In order to determine the exact concentrations of inhibitors required for complete inhibition of each OXPHOS complex, we used the same amount of cells as used for spectrofluorometry to measure oxygen consumption with a Clark-type electrode (Oxytherm System, Hansatech, UK). Intact cell respiration was recorded from cells suspended in 500 µL of Respiraton Buffer A (225 mM sucrose, 75 mM mannitol, 10 mM Tris/HCl, 10 mM KCl, 10 mM KH2PO4, 5 mM MgCl2, pH 7.4) plus 10 mg/mL freshly added BSA, permeabilised by the addition of 80 µg/mL digitonin at 37°C. Substrate concentrations were as follows: 10 mM ADP, 5 mM pyruvate and malate (cI+cIII), 10 mM succinate (cII+cIII), 50 µM TMPD/1 mM ascorbate (cIV). The required concentrations for full inhibition were determined to be 3 µM rotenone (cI inhibitor), 1 µM antimycin (cIII inhibitor), 0.8 mM potassium cyanide (KCN, cIV inhibitor), 5 µM oligomycin (cV inhibitor), and 100 µM nPG (AOX inhibitor).
10
Mitochondrial Respiration in HL-1 Cells
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After glucose treatments during 12 or 72h, HL-1 cells were harvested and centrifuged for 2 min at 300 g. The oxygen consumption was measured at 37°C on 0.2% (w/v) digitonin-permeabilized cells using a Clark-type oxygen electrode (Oxytherm system, Hansatech Instruments, Cergy Saint Christophe, France). Pellets were resuspended in the respiration buffer (in mmol/l: K-MES 100, KH2PO4 5, EGTA 1, EDTA 3, ADP 1 and bovine serum albumin 1 mg/ml, pH 7.4), then cells were placed in the incubation chamber. Several substrates were used: 25 mmol/l succinate + 25 μmol/l rotenone, 80 μmol/l palmitate + 5 mmol/l malate or 10 mmol/l pyruvate + 5 mmol/l malate. Respiration rate was measured using the Oxygraph Plus software, and results were expressed as nmol O2.min-1.mg-1 of protein.
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