Cells were collected at each time point, and measurements were made directly on 1 mL of the cell suspensions. Oxygen evolution rates (gross oxygen evolution) were measured using Clark Electrode (Hansatech Oxygraph). The assayed actinic light steps included 6, 14, 30, 45, 68, 103, 176, 301, 571, 1024, and 1931 μmol.m−2 s−1. Oxygen evolution rates were determined based on the sum of the net oxygen evolution rates over the last 45 seconds of each light step and the absolute value of the dark respiration rates.
Clark electrode
Manufactured by Hansatech
Sourced in United Kingdom
The Clark electrode is a type of oxygen sensor used to measure the concentration of dissolved oxygen in a solution. It functions by using an electrochemical reaction to detect the presence of oxygen molecules. The core function of the Clark electrode is to provide a quantitative measurement of the oxygen content in a sample.
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Lab products found in correlation
Oxytherm, by Hansatech (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
L450p1600mm, by Thorlabs (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Biochemical kits, by Merck Group (1 mentions)
Atp measurement kit, by Thermo Fisher Scientific (1 mentions)
25 protocols using clark electrode
1
Oxygenic Photosynthesis Kinetics
2
Photosynthetic oxygen evolution analysis
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For comparison with the photocurrent measurements the O2 evolution rate was measured in a Clark electrode (Hansatech Instruments, UK) with 0.1 mg Chl membranes and 3 mM Fe(III)CN as an electron acceptor. The sample was illuminated with a solar simulator (model 10500 solar simulator, ABET Technologies, USA) at 1 Sun for 10 min. For other measurements the O2 evolution rate was calculated for 0.1 mg Chl with 3 mM DCBQ, Fe(III)CN or 1,1′-dimethyl-4,4′-bipyridinium (MV, Aldrich) as electron acceptors with or without DCMU and DBMIB, under white light illumination (MRC). Reduced MV reduces the O2 and therefore it measures the consumption rate of O2.
3
Cellular Respiration in Glucose and Lactate
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Cells were grown as indicated, cultured up to the mid-log phase in the presence of 2% glucose, normalized and then shifted to 2% lactic acid-containing medium for 3 h at 30°C. After incubation cells were centrifuged at 5000 g for 10 min, and cell pellets were suspended either in 2% glucose or 2% lactate medium at a concentration of 108 cells/ml. Cellular OCR was measured using an Oxytherm fitted with a Clark electrode (Hansatech, UK). Respiration was measured for 4 min and then blocked with the CIV-specific inhibitor, sodium azide, at 1 mM concentration.
4
Quantifying Photocatalytic Water Oxidation
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The photocatalytic water oxidation activity of Ir-DHC on different supports was quantified by monitoring the rate of O2 evolution with a Clark electrode (Hansatech).20 (link) Light-driven water oxidation was performed in a buffered aqueous solution at pH 5.7 (NaHCO3 (28 mM), Na2SiF6 (22 mM), and 50 mM Na2SO4). Na2S2O8 (20 mM) served as a sacrificial electron acceptor. [Ru(bpy)3]Cl2 (2.7 mM) was employed as the photosensitizer.
1.5 mL of a freshly prepared catalyst suspension (10 mg mL−1) was added to a cylindrical quartz vessel (diameter of 1 cm), whose temperature was controlled by a water-jacket. Under stirring (150 rotations per min), the suspension was allowed to equilibrate to the desired temperature for 10 min. Then, Ar was bubbled through the suspension for 3 min to remove dissolved O2. After a steady baseline in O2 concentration was achieved as monitored with the Clark electrode, the cell was sealed with a rubber stopper. Water oxidation was initiated with a 1 s light pulse (beam diameter = 7 mm (1/e2 value); wavelength = 450 nm) from a laser diode (Thorlabs, model number: L450P1600MM). The ensuing evolution of O2 was monitored over 1.5 min. The initial rate of O2 evolution was obtained by fitting a linear function to the initial rise of the O2 concentration.
1.5 mL of a freshly prepared catalyst suspension (10 mg mL−1) was added to a cylindrical quartz vessel (diameter of 1 cm), whose temperature was controlled by a water-jacket. Under stirring (150 rotations per min), the suspension was allowed to equilibrate to the desired temperature for 10 min. Then, Ar was bubbled through the suspension for 3 min to remove dissolved O2. After a steady baseline in O2 concentration was achieved as monitored with the Clark electrode, the cell was sealed with a rubber stopper. Water oxidation was initiated with a 1 s light pulse (beam diameter = 7 mm (1/e2 value); wavelength = 450 nm) from a laser diode (Thorlabs, model number: L450P1600MM). The ensuing evolution of O2 was monitored over 1.5 min. The initial rate of O2 evolution was obtained by fitting a linear function to the initial rise of the O2 concentration.
5
Measuring O2 Consumption in Leishmania
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Variation in the O2 consumption rate of L. donovani promastigotes was measured in a Clark electrode (Hansatech Instruments, King’s Lynn, UK) at 1 × 108 cells/mL in 600 μL of respiration buffer (10 mM Tris-HCl, 125 mM sucrose, 65 mM KCl, 1 mM MgCl2, 2.5 mM NaH2PO4, 0.3 mM EGTA, 5 mM succinic acid; pH 7.2). Compounds were added at 100 × final concentration to whole parasites, and oxygen consumption was monitored for 8–10 min.
6
Cucumber Leaf Respiration Measurement
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Respiration rate was measured as a decrease in O2 concentration using a Clark electrode (Hansatech Instruments Ltd., Norfolk, UK) in a LD/2 chamber connected to a data reader device CB1D (Hansatech). Measurements were made on discs (diameter of 5 cm), cut from cucumber leaves, under 21% O2, 350 ppm CO2 in a closed system, at a temperature of 25 °C.
7
Measuring Oxygen Consumption in RAW264.7 Cells
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To measure oxygen consumption, RAW264.7 cells (ATCC TIB-71) were cultured in Dulbecco's modified Eagle's medium with 4.5 g/l D-glucose and L-glutamine (Gibco, Thermo Fisher Scientific, NH, USA) supplemented with 10% of heat-inactivated fetal bovine serum (Gibco, Thermo Fisher Scientific, NH, USA) and 100 U/ml penicillin-streptomycin (Thermo Fisher Scientific, NH, USA) at 37°C with 5% CO2. The cells were primed either with 50 ng/ml of LPS or HKLm to correspond to an MOI of 530. At 19-24 h later the media was changed to fresh media including priming agents. A minimum of 30 min later the oxygen consumption from 5 million cells was measured at 37°C in their culture media using a Clark electrode (Hansatech, UK). Mitochondrial respiration was measured as the total minus the background oxygen consumption; the latter being determined by exposing the cell suspension to 150-270 nM of antimycin A (Sigma-Aldrich, MO, USA), a potent inhibitor of the respiratory chain complex III.
8
Photosynthesis Rate Measurement Protocol
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Out of the possible ways of measuring the photosynthesis rate, we consider measurements of the maximal rate of photosynthetic oxygen evolution at a specific actinic light using a Clark electrode (Hansatech, UK). In order to use PI measurements for parameter estimation, knowledge of the exact experimental light protocol is paramount, as established in Bernardi et al. [22 (link)]. Here, we consider PI measurements obtained from three separate experiments. Each experiment, involves exposing a dark adapted sample to two different light intensities over variable time periods (the exact protocols are reported in Table 2 ). The photosynthesis rate is measured at the end of each time period, thus providing a total of six experimental PI points. Sample 1 is to be included in the calibration data set, whereas the other two experiments are used for model validation only.
9
Cloning and expression of NrdA and NrdB
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All chemicals were purchased from Sigma-Aldrich or VWR and used as received unless otherwise stated. Cloning of the N-terminal hexa-histidine tag containing pET-nrdA, pET-nrdB, pET-nrdB∆169 and pET-NrdB∆Grx coding for F. ignava NrdA, NrdB, NrdB∆169 and NrdB∆Grx proteins was done as described in [12 (link)]. Cloning of pET-nrdB∆99 encoding L. blandensis NrdB∆99 (LbNrdB∆99) was done as described in [11 ]. All anaerobic work was performed in an MBRAUN glovebox ([O2] < 10 ppm). Oxygen detection was done using a standard Clark electrode (Hansatech Instruments), separated from the sample solution by a Teflon membrane, and the signal was recorded using the CalMeter software package by Calmetric. Air-saturated water solutions ([O2]25 °C = 253 µM) were used for the calibration of the electrode.
10
Oxygen Consumption Measurement in Cells
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Oxygen consumption was measured by placing 2 × 106 cells into a closed chamber equipped with a Clark electrode (Hansatech, Cambridge, UK) as described previously [27 (link)]. Data were the averages of samples ± SE from three (5 min and 5 h) or six (24 h) independent experiments.
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