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Two chamber oxygraph

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The Two-chamber Oxygraph is a laboratory instrument designed to measure oxygen consumption in biological samples. The device features two independent chambers, allowing for simultaneous analysis of two samples. The core function of the Two-chamber Oxygraph is to accurately monitor and record the changes in oxygen levels within the chambers over time.

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Lab products found in correlation

Datlab software, by Oroboros (1 mentions)

3 protocols using two chamber oxygraph

1

Cellular Oxygen Consumption Analysis

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Culture medium was collected from cells treated with either RPMI or β-glucan (1 μg/mL or 10 μg/mL). After stimulation, the cells were trypsinized, washed, and resuspended in the collected culture medium. Cell suspensions containing 1 × 106 cells were then used for cellular O2 consumption analysis. Oxygen consumption was measured at 37°C using polarographic oxygen sensors in a two-chamber Oxygraph (OROBOROS Instruments, Innsbruck, Austria). DatLab software (Oroboros) was used for data acquisition (2 s time interval) and analysis (Gnaiger, 2001 (link)). First, basal respiration (baseline oxygen consumption) was measured. Next, leak respiration was determined by addition of 2.5 μM of the specific complex V inhibitor oligomycin A (OLI). Then, maximal electron transport chain complex (ETC) capacity (maximum oxygen consumption) was quantified by applying increasing concentrations of the mitochondrial uncoupler FCCP (0.25 to 20 μM final maximal concentration). Finally, minimal (non-mitochondrial) respiration was assessed by addition of the specific complex I inhibitor rotenone (ROT; 100 nM) and the complex III inhibitor antimycin A (AA; 2.5 μM).
Keating S.T., Groh L., van der Heijden C.D., Rodriguez H., dos Santos J.C., Fanucchi S., Okabe J., Kaipananickal H., van Puffelen J.H., Helder L., Noz M.P., Matzaraki V., Li Y., de Bree L.C., Koeken V.A., Moorlag S.J., Mourits V.P., Domínguez-Andrés J., Oosting M., Bulthuis E.P., Koopman W.J., Mhlanga M., El-Osta A., Joosten L.A., Netea M.G, & Riksen N.P. (2020). The Set7 Lysine Methyltransferase Regulates Plasticity in Oxidative Phosphorylation Necessary for Trained Immunity Induced by β-Glucan. Cell Reports, 31(3), 107548.
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2

Measuring Cellular Oxygen Consumption

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The culture medium was collected and the BRAF mutant melanoma cells were trypsinized, washed, and resuspended to approximately 2 × 10E6 cells per 2 ml in the collected culture medium and used to measure cellular oxygen consumption. Oxygen consumption was measured at 37 °C using polarographic oxygen sensors in a two-chamber Oxygraph (Oroboros Instruments, Innsbruck, Austria) as previously described [26 (link)]. Briefly, cells were first allowed to respire at basal level for 10 min and then inhibited acutely by a stepwise addition of increasing concentrations (0–100 nM) of BAY 87-2243 until respiration was maximally blocked. Basal (routine) respiration was set at 100 % to which all other data points were related.
Schöckel L., Glasauer A., Basit F., Bitschar K., Truong H., Erdmann G., Algire C., Hägebarth A., Willems P.H., Kopitz C., Koopman W.J, & Héroult M. (2015). Targeting mitochondrial complex I using BAY 87-2243 reduces melanoma tumor growth. Cancer & Metabolism, 3, 11.
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3

Mitochondrial Respiration Measurement

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Oxygen consumption was measured in freshly isolated heart mitochondria at 37°C using polarographic oxygen sensors in a two-chamber Oxygraph (OROBOROS Instruments) as previously described [4 (link)]. Oxygen consumption was measured with specific substrates for complex I (2 mM malate, 5 mM glutamate) or complex II (10 mM succinate, 5 mM glutamate) in MS-EGTA buffer (210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 5 mM HEPES) with 2 mM H2KPO4 and 1 mM MgCl2.
Anderson C.J., Bredvik K., Burstein S.R., Davis C., Meadows S.M., Dash J., Case L., Milner T.A., Kawamata H., Zuberi A., Piersigilli A., Lutz C, & Manfredi G. (2019). ALS/FTD mutant CHCHD10 mice reveal a tissue-specific toxic gain-of-function and mitochondrial stress response. Acta neuropathologica, 138(1), 103-121.
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