Analysis of the effects of herbicides or inhibitors on the kinetics of charge recombination was performed using a Cary60 spectrophotometer connected to an external 1 cm cuvette holder (Ocean Optics) via a fibre-optic coupler. A white light pulse of 50 ms duration was applied to the sample cuvette at 90° to the pulsed measuring beam by an HL-2000-FHSA white light source with a fast shutter (Ocean Optics) delivering approximately 25 W m−2 illumination at the cuvette surface. The actinic pulse was triggered by a TGP110 pulse generator (Thurlby Thandur Instruments). Solutions of 13.2 µM reaction centres in 20 mM Tris (pH 8.0)/ 30 µM UQ0 were prepared with and without the addition of 500 µM atrazine, terbutryn, stigmatellin, bromoxynil, bentazon, capsaicin or DCMU. Test samples were loaded into to a 3×3 mm fluorescence cuvette (Hellma) aligned to the measuring and excitation pulses and absorbance at 865 nm was measured for 20 s after delivery of the light pulse, with a further period of at least 60 s to allow full dark adaptation of the reaction centres before re-excitation. A set of eight kinetic traces were recorded for each sample, averaged, and fitted using a single or double exponential function in Origin 8 (OriginLab).
Fluorescence cuvette
Manufactured by Hellma
Sourced in Japan
The Fluorescence cuvette is a laboratory equipment designed for the measurement of fluorescence properties of samples. It provides a standardized container to hold the sample during fluorescence analysis.
Automatically generated - may contain errors
Lab products found in correlation
External 1 cm cuvette holder, by OceanOptics (2 mentions)
Cary60 spectrophotometer, by OceanOptics (2 mentions)
Hl 2000 fhsa, by OceanOptics (2 mentions)
Origin 8, by OriginLab (2 mentions)
F 7000 spectrofluorometer, by Hitachi (1 mentions)
Invia spectrometer, by Renishaw (1 mentions)
Vertex 70v spectrometer, by Bruker (1 mentions)
Invia raman microscope, by Renishaw (1 mentions)
Uh4150 spectrometer, by Hitachi (1 mentions)
Fp 8200, by Jasco (1 mentions)
Prism 7, by GraphPad (1 mentions)
7 protocols using fluorescence cuvette
1
Spectrophotometric Analysis of Herbicide Impacts
2
Fluorescence Emission Spectra Measurement
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Room temperature fluorescence emission spectra were recorded with a F-7000 spectrofluorometer (Hitachi, Tokyo, Japan) using a fluorescence cuvette with the dimensions of 5 × 5 mm2 (Hellma, Müllheim, Germany). The excitation was set at 434 nm. The excitation and emission slits were 5 nm.
3
Spectrophotometric Analysis of Herbicide Impacts
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Analysis of the effects of herbicides or inhibitors on the kinetics of charge recombination was performed using a Cary60 spectrophotometer connected to an external 1 cm cuvette holder (Ocean Optics) via a fibre-optic coupler. A white light pulse of 50 ms duration was applied to the sample cuvette at 90° to the pulsed measuring beam by an HL-2000-FHSA white light source with a fast shutter (Ocean Optics) delivering approximately 25 W m−2 illumination at the cuvette surface. The actinic pulse was triggered by a TGP110 pulse generator (Thurlby Thandur Instruments). Solutions of 13.2 µM reaction centres in 20 mM Tris (pH 8.0)/ 30 µM UQ0 were prepared with and without the addition of 500 µM atrazine, terbutryn, stigmatellin, bromoxynil, bentazon, capsaicin or DCMU. Test samples were loaded into to a 3×3 mm fluorescence cuvette (Hellma) aligned to the measuring and excitation pulses and absorbance at 865 nm was measured for 20 s after delivery of the light pulse, with a further period of at least 60 s to allow full dark adaptation of the reaction centres before re-excitation. A set of eight kinetic traces were recorded for each sample, averaged, and fitted using a single or double exponential function in Origin 8 (OriginLab).
4
Spectroscopic Characterization of N-methylacetohydroxamic Acid
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All solvents and analytical-grade chemicals were obtained from commercial suppliers and used without further purification. N-methylacetohydroxamic acid (NMAH) was prepared following the improved procedure described recently by Brandès et al. 3 The sample used herein was taken from the same batch for which analytical data ( 1 H and 13 C NMR, CHN contents) have been reported elsewhere. 3 An aqueous stock solution of in the mother solution.
Fourier-transform mid-infrared (400-4000 cm -1 ) spectra (FTMIR) were recorded at 4 cm -1 resolution on a Bruker VERTEX 70v spectrometer fitted with an A225 diamond attenuated total reflection (ATR) accessory (Bruker) and a DTGS (deuterated triglycine sulfate) detector (350-4000 cm -1 ). Raman spectra were collected with a Renishaw inVia spectrometer equipped with a 632.8 nm He-Ne laser excitation source, a 1800 grooves/mm grating, and a microscope fitted with a either a 50 (solid samples) or 20 (liquid samples)
objective. Solids were deposited on a glass slide, while solutions were introduced in a fluorescence cuvette of 1 cm path length (Hellma). Wavenumbers were calibrated with respect to the silicon scattering line at 520(1) cm
Fourier-transform mid-infrared (400-4000 cm -1 ) spectra (FTMIR) were recorded at 4 cm -1 resolution on a Bruker VERTEX 70v spectrometer fitted with an A225 diamond attenuated total reflection (ATR) accessory (Bruker) and a DTGS (deuterated triglycine sulfate) detector (350-4000 cm -1 ). Raman spectra were collected with a Renishaw inVia spectrometer equipped with a 632.8 nm He-Ne laser excitation source, a 1800 grooves/mm grating, and a microscope fitted with a either a 50 (solid samples) or 20 (liquid samples)
objective. Solids were deposited on a glass slide, while solutions were introduced in a fluorescence cuvette of 1 cm path length (Hellma). Wavenumbers were calibrated with respect to the silicon scattering line at 520(1) cm
5
Intracellular Fluorescent Nanodiamonds Characterization
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If not stated otherwise, PL spectra were recorded using a Renishaw InVia Raman microscope; the excitation wavelengths were 514 nm and 488 nm with 15 mW laser power, using 50× long infinity corrected distance objective (numerical aperture 0.4). The measurements in aqueous solution (0.2 mg ml -1 ) and in cell medium (the same type as used for cell incubation) were performed in a Hellma fluorescence cuvette (type no. 105.252-QS). For cellular measurements, FND, FND-PEI or FND-PEI-DNA (final concentration 25 µg ml -1 ) were incubated with cells for 30, 60 and 120 min, washed with PBS (to remove efficiently the particles localized extracellularly), fixed in ethanol and stored at -20 °C before measurements. Measurements were performed from intracellular regions on 10 cells for each sample. Data show average of 10 measure-ments after background subtraction. All the measurements were performed using a 50× long distance objective.
6
Ultraviolet Absorption Spectroscopy Protocol
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All of the ultraviolet absorption spectra were recorded on a Hitachi UH4150 spectrometer with a water circulation temperature controller. All the ultraviolet absorption spectra data were collected at the same sample concentration at room temperature (30 μl sample was diluted into 3 ml solvent), if not otherwise specified. For ultraviolet absorption study, Hellma fluorescence cuvettes were used (standard cells). The data collection interval is 1 nm and the scan rate is 800 nm·min−1. The light path was 10 mm (macro, suprasil quartz, limit 200–2,500 nm spectral range, path length 10 × 10 mm, chamber volume 3,500 μl). The background was corrected with the solvents used. Solvents for measurements at high temperatures were pre-heated before diluting the samples. It is of help to point out that the absorbance units are given as a.u. in our study; still, it is possible to compare the absorbance in a relative fashion for the various reactions presented in one figure, such as Fig. 2 .
7
Kinetic Analysis of Ubiquitin Hydrolysis by USP25
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Ub-AMC hydrolysis assays and kinetic analysis were set up in fluorescence cuvettes (Hellma Analytics) in 100 μl buffer with 150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 5 mM DTT, 0.1% (v v−1) Tween-20 and measured with a fluorescence spectrometer (JASCO FP-8200) at excitation and emission wavelengths of 355 and 455 nM. Hydrolysis assays were performed at 37 °C with 200 nM USP25 constructs and 0.1 μM Ub-AMC in triplicates. Kinetic analysis (Michaelis–Menten kinetic measurements) were carried out using 5 nM USP25 constructs with a series of Ub-AMC substrate titrations at 37 °C. Initial rates of substrate hydrolysis were obtained using linear regression. Kinetic curves were obtained by plotting the measured enzyme initial rates vs. the corresponding substrate concentrations, followed by the modeling using nonlinear regression fit with Michaelis–Menten equation. All data were processed using GraphPad Prism 7.0.
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