Determination of COX- and AOX-dependent oxygen consumption was performed using strains cultivated on M2 medium for 2 days and in CM liquid medium for 2 days, as described above. Small pieces of mycelium were subsequently transferred into the high-resolution respirometer (Oxygraph-2k series C and G, Oroboros Instruments, Innsbruck, Austria) and oxygen consumption was measured in liquid CM medium according to the manufacturer’s instructions. Then, 1 mM potassium cyanide (KCN; Fluka, Buchs, Switzerland; 60178) was added to inhibit respiration via COX, and 4 mM salicylhydroxamic acid (SHAM; Sigma-Aldrich, St. Louis, MO, USA; S607) was added to inhibit respiration via AOX. Data were analyzed using the manufacturer’s software DatLab 6.
Oxygraph 2k series c and g
Manufactured by Oroboros
Sourced in Austria
The Oxygraph-2k series C and G are high-performance laboratory equipment designed for the precise measurement of oxygen concentration and respiration rates. These instruments utilize a polarographic oxygen sensor to accurately detect and monitor oxygen levels in a variety of biological and chemical samples. The Oxygraph-2k series C and G provide reliable and consistent data, making them a valuable tool for researchers and scientists in various fields.
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4 protocols using oxygraph 2k series c and g
1
Measuring COX and AOX Respiration
2
Mitochondrial Respiration Profiling
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For measurement of the mitochondrial oxygen consumption rate, 150 μg of freshly isolated mitochondria was used and measured by high-resolution respirometry at 27 °C (Oxygraph-2k series C and G, OROBOROS Instruments, Innsbruck, Austria). Mitochondria were injected into a chamber with 2 ml of air-saturated buffer (0.3 M sucrose, 10 mM KH2PO4, 5 mM MgCl2, 1 mM EGTA, 10 mM KCl and 0.1% BSA; pH 7.2) in presence vs. absence of 1 μM SM19. To stimulate complex I-dependent phosphorylating respiration in the presence of ADP, 10 mM pyruvate (Sigma-Aldrich, P2256), 2 mM malate (Sigma-Aldrich, M1000) and 1.5 mM ADP (Sigma-Aldrich, A5285) was added.99 (link) When oxygen consumption reached a constant level, 1 mM potassium cyanide (KCN, inhibitor of cytochrome c oxidase (COX)) and 1 mM salicylhydroxamic acid (SHAM, inhibitor of the alternative oxidase (AOX)) were added to completely block respiration. Wild-type and ΔPaCrd1 mitochondria were analyzed in presence of absence of 1 μM SM19. For analyzing the data, the manufacturer's software DatLab 6 was used.
3
Oxygen Consumption Rate Measurement
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The measurement of oxygen consumption rate was performed at 27°C by high-resolution respirometry (Oxygraph-2k series C and G, OROBOROS Instruments, Innsbruck, Austria). Cultures derived from mononucleate ascospores of wild type, ΔPaSnf1, ΔPaClpP, and ΔPaSnf1/ΔPaClpP were grown on M2 plates covered with a cellophane foil for 2 days, shifted into CM liquid medium, and further incubated for 3 days at 27°C under shaking and constant light. A small piece of mycelium was placed into the Oroboros respirometer chamber into 2 ml fresh CM media. The oxygen consumption rate was determined as previously described in Fischer et al. (2015a) (link). After the measurement, the mycelium was placed into a 2-ml tube and boiled for 10 min at 95°C. Subsequently, the mycelium was air-dried for 2 days, and dry weight was determined. Finally, basal oxygen consumption per mg mycelium was calculated. For the analysis of the data, the DatLab6 software from Oroboros (Innsbruck, Austria) was used.
4
Mitochondrial Respiration by Oxygraph
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Determination of mitochondrial oxygen consumption was performed at 27°C by high-resolution respirometry (Oxygraph-2k series C and G, OROBOROS Instruments, Innsbruck, Austria). 200 μg freshly prepared mitochondria was injected into 2 ml air saturated oxygen buffer (0.3 M sucrose, 10 mM KH2PO4, 5 mM MgCl2, 1 mM EDTA, 10 mM KCl, and 0.1% BSA; pH 7.2). To promote the ADP-limited complex I-dependent state 4 respiration (state 4) 10 mM pyruvate (Sigma-Aldrich, P2256) and 2 mM malate (Sigma-Aldrich, M1000) were added. Subsequently, 1.5 mM ADP (Sigma-Aldrich, A5285) was added to determine complex I-dependent state 3 respiration (state 3). Data were analyzed using the manufacturer’s software DatLab 6.
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