α-Lipoamide dehydrogenase activity of rat liver mitochondria was analyzed, probing the reversed reaction of the enzyme [39 (link)]. After 3 min incubation of rat liver mitochondria (0.3 mg protein/mL) in MTP medium in the presence of 5 mM MgCl2, 1 mM ATP, and the respective amounts of VPA (or its derivatives) in 1 mL fluorimetric cuvette, the following additions were subsequently made: 5 mM malate and 10 mM pyruvate, 6.7 µM rotenone, 4 µM KCN, 75 µM α-lipoamide (30 mM stock solution was dissolved in 50% alcohol), and 10 mM arsenite, 0.4 µM 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole (TTFB). The NAD(P)H reduction was monitored at λex = 340 nm, λem = 450 nm in a Shimadzu RF-5001PC (Shimadzu Scientific, Columbia, MD, USA) spectrofluorophotometer. DLDH activity was estimated from the NAD(P)H fluorescence increase after the addition of α-lipoamide. In agreement with Luis et al. [11 (link)], complete inhibition of DLDH activity was observed after the application of 10 mM arsenite.
Rf 5001pc
Manufactured by Shimadzu
Sourced in Japan
The RF-5001PC is a fluorescence spectrofluorophotometer manufactured by Shimadzu. It is designed to measure the fluorescence intensity of liquid samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanoscope multimode, by Veeco (2 mentions)
Fura 2 am, by Thermo Fisher Scientific (2 mentions)
Vertex v70, by Bruker (1 mentions)
Nucleotides, by Merck Group (1 mentions)
Ar c 118925xx, by Bio-Techne (1 mentions)
Carboxy h2dcfda dye, by Thermo Fisher Scientific (1 mentions)
Zoe fluorescent cell imager, by Bio-Rad (1 mentions)
7 protocols using rf 5001pc
1
Fluorometric Assay of Lipoamide Dehydrogenase Activity
2
Fluorescence Spectra Measurement at 23.5-18.0 °C
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The fluorescence
spectra were measured with a fluorescence spectrometer RF-5001 PC
(Shimadzu, Japan). The emission spectra were recorded in the range
of 250–600 nm at an excitation wavelength of 295 nm. The fluorescence
spectra were measured at the temperature range of 23.5–18.0
°C.
spectra were measured with a fluorescence spectrometer RF-5001 PC
(Shimadzu, Japan). The emission spectra were recorded in the range
of 250–600 nm at an excitation wavelength of 295 nm. The fluorescence
spectra were measured at the temperature range of 23.5–18.0
°C.
3
Characterization of Nitrogen-Doped Carbon Quantum Dots
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Elemental analysis of prepared N-CQD samples was performed by energy-dispersive X-ray method (EDX). The equipment used for the EDX analysis was LEO Electron Microscopy Ltd., England, model 1430 VP.
The High Resolution Transmission Electron Microscopy (HRTEM) images were taken by using a transmission electron microscope F20X-TWIN (FEI-Tecnai, Norcross, GA, USA) operated at 200 kV. The drop of sample solution was placed on a Cu-grid coated with an ultrathin amorphous carbon film and then dried under ambient conditions.
The morphology of N-CQD dispersed on Si wafers was analyzed at room temperature, in air, using a microscope with a scanning SPM probe of the NanoScope MultiMode type (Veeco Metrology, Inc., Santa Barbara, CA, USA) which operated in a tapping mode.
Fourier-transform infrared (FTIR) spectroscopy data were acquired by using a Vertex V70 (Bruker Optic), in ATR mode techniques (single reflection using diamond crystal), in the frequency range 6000–15 cm−1.
The fluorescence spectra were measured with a fluorescence spectrometer RF-5001PC (Shimadzu, Japan). Excitation maximum was experimentally established at 360 nm. Fluorescence decay curves and photoluminescence (PL) absolute quantum yield (QY) for as-obtained N-CQDs were measured at room temperature. UV–Vis spectra of N-CQDs were acquired by using a Jasco 660 spectrometer in the range of 200–800 nm.
The High Resolution Transmission Electron Microscopy (HRTEM) images were taken by using a transmission electron microscope F20X-TWIN (FEI-Tecnai, Norcross, GA, USA) operated at 200 kV. The drop of sample solution was placed on a Cu-grid coated with an ultrathin amorphous carbon film and then dried under ambient conditions.
The morphology of N-CQD dispersed on Si wafers was analyzed at room temperature, in air, using a microscope with a scanning SPM probe of the NanoScope MultiMode type (Veeco Metrology, Inc., Santa Barbara, CA, USA) which operated in a tapping mode.
Fourier-transform infrared (FTIR) spectroscopy data were acquired by using a Vertex V70 (Bruker Optic), in ATR mode techniques (single reflection using diamond crystal), in the frequency range 6000–15 cm−1.
The fluorescence spectra were measured with a fluorescence spectrometer RF-5001PC (Shimadzu, Japan). Excitation maximum was experimentally established at 360 nm. Fluorescence decay curves and photoluminescence (PL) absolute quantum yield (QY) for as-obtained N-CQDs were measured at room temperature. UV–Vis spectra of N-CQDs were acquired by using a Jasco 660 spectrometer in the range of 200–800 nm.
4
Physicochemical Characterization of N-CQDs
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The full physicochemical characterization was performed and described recently [8 (link),27 (link)]. These analyses concern: the effect of purification, and the elemental analysis of the prepared N-CQDs samples performed by the energy dispersive X-ray method (EDX), AFM, and HRTEM.
The equipment used for the EDX analysis was the LEO Electron Microscopy Ltd., Cambridge, England, model 1430 VP.
The High-Resolution Transmission Electron Microscopy (HRTEM) images (Figure S7 ) were taken using a transmission electron microscope, F20X-TWIN (FEI-Tecnai), operated at 200 kV. The drop of sample solution was placed on a Cu grid coated with an ultrathin amorphous carbon film and then dried under ambient conditions.
The morphology of the N-CQDs dispersed on Si wafers was analyzed at room temperature in the air using a microscope with a scanning SPM probe of the NanoScope MultiMode type (Veeco Metrology, Inc., Santa Barbara, CA, USA), which operated in a tapping mode.
The fluorescence spectra were measured with a fluorescence spectrometer, RF-5001PC (Shimadzu, Japan). The excitation maximum was experimentally established at 360 nm. The UV-vis spectra of the N-CQDs were acquired using a Jasco 660 spectrometer in a range 200–800 nm.
Presented here, the SEM pictures show the N-CQDs immobilized on glass beads (Figure S8 ).
The equipment used for the EDX analysis was the LEO Electron Microscopy Ltd., Cambridge, England, model 1430 VP.
The High-Resolution Transmission Electron Microscopy (HRTEM) images (
The morphology of the N-CQDs dispersed on Si wafers was analyzed at room temperature in the air using a microscope with a scanning SPM probe of the NanoScope MultiMode type (Veeco Metrology, Inc., Santa Barbara, CA, USA), which operated in a tapping mode.
The fluorescence spectra were measured with a fluorescence spectrometer, RF-5001PC (Shimadzu, Japan). The excitation maximum was experimentally established at 360 nm. The UV-vis spectra of the N-CQDs were acquired using a Jasco 660 spectrometer in a range 200–800 nm.
Presented here, the SEM pictures show the N-CQDs immobilized on glass beads (
5
Fura-2 AM-Based Calcium Imaging in Myoblasts
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Myoblasts were cultured on glass coverslips in 35 mm diameter dishes for 48 h under the conditions described above. Cells (70–80% confluent) were loaded with 2 μM Fura 2 AM (MolecularProbes, Oregon) in the serum-free culture medium for 20 min at 37 °C in a humidified atmosphere of 95% O2 and 5% CO2. The cells were then washed twice with the solution composed of 5 mM KCl, 1 mM MgCl2, 0.5 mM Na2HPO4, 25 mM HEPES, 130 mM NaCl, 1 mM pyruvate, 5 mM d -glucose, and 0.1 mM CaCl2, pH 7.4 and the coverslips were mounted in a cuvette containing 3 ml of the nominally Ca2+-free assay solution (as above but 0.1 mM CaCl2 was replaced by 0.05 mM EGTA) and placed (at RT) in a spectrofluorimeter (Shimadzu, RF5001PC). The cells were treated with nucleotides (Sigma) applied at a concentration indicated in the relevant figures and 10 μM AR-C 118925XX (216657-60-2, TOCRIS Bioscience) applied 10 min before the addition of agonists. Fluorescence was recorded at 510 nm, with excitation at 340 and 380 nm. At the end of each experiment, the Fura 2 fluorescence was calibrated by the addition of 33 μM ionomycin to determine maximal fluorescence, followed by the addition of EGTA to complete the removal of Ca2+. Cytosolic Ca2+ concentration [Ca2+]c was calculated according to Grynkiewicz et al.38 (link).
6
Measuring Cytosolic Ca2+ in Myoblasts
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Myoblasts were cultured on glass coverslips in a 6-well plate (500 000 cells/well) for 48 h under conditions described above. Cells (70-80% confluent) were loaded with 1 M Fura 2 AM (Molecular Probes, Oregon) in culture medium for 15 min at 33°C in a humidified atmosphere of 95% O 2 and 5% CO 2 . The cells were then washed twice with the solution composed of 5 mM KCl, 1 mM MgCl 2 , 0.5 mM Na 2 HPO 4 , 25 mM HEPES, 130 mM NaCl, 1 mM pyruvate, 5 mM D-glucose, and 0.1 mM CaCl 2 , pH 7.4 and the coverslips were mounted in a cuvette containing 3 ml of either the nominally Ca 2+ -free assay solution (as above but 0.1 mM CaCl 2 was replaced by 0.05 mM EGTA) or Ca 2+ -containing assay solution (as above but with 2 mM CaCl 2 ) and maintained at RT in a spectrofluorimeter (Shimadzu, RF5001PC).
Fluorescence was recorded at 510 nm with excitation at 340 and 380 nm. At the end of each experiment the Fura 2 fluorescence was calibrated by addition of 8 M ionomycin to determine maximal fluorescence followed by addition of EGTA to complete removal of Ca 2+ . Cytosolic Ca 2+ concentration [Ca 2+ ] c was calculated according to Grynkiewicz et al. [35] . The cells were treated with 500 µM and 1 mM ATP, 100 µM and 500 µM UTP (both Sigma) and 10 µM AR-C 118925XX (216657-60-2, TOCRIS Bioscience) applied 10 min prior to the addition of ATP and UTP. Each experiment was repeated at least 5 times.
Fluorescence was recorded at 510 nm with excitation at 340 and 380 nm. At the end of each experiment the Fura 2 fluorescence was calibrated by addition of 8 M ionomycin to determine maximal fluorescence followed by addition of EGTA to complete removal of Ca 2+ . Cytosolic Ca 2+ concentration [Ca 2+ ] c was calculated according to Grynkiewicz et al. [35] . The cells were treated with 500 µM and 1 mM ATP, 100 µM and 500 µM UTP (both Sigma) and 10 µM AR-C 118925XX (216657-60-2, TOCRIS Bioscience) applied 10 min prior to the addition of ATP and UTP. Each experiment was repeated at least 5 times.
7
Cobalt-induced ROS Measurement Protocol
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Measurement of ROS activity was performed using carboxy-H2DCFDA dye (Invitrogen, Waltham, MA, USA, UK C400, Lot number 28351W) as described earlier [16 (link),17 (link)]. Cells were seeded at 5 × 104 cells/ mL and incubated at 37 °C and 5% CO2/air for two days. The cells were then treated with a range of cobalt concentrations (0, 100, 150, and 200 μM) for two periods of time (4 and 24 h). Then the medium was removed from all wells, and the cells were washed twice with DPBS to remove any traces of media from the wells. The addition of 200 μL of carboxy-H2DCFDA at 25 μM in DPBS followed the washing step. The culture was incubated in the dark at 37 °C and 5% CO2 air for 30 min. Cells were washed twice with DPBS after incubation. Cells were examined by microscope (ZOE Fluorescent Cell Imager, BIO-RAD, Singapore) and pictures were taken immediately. Finally, triton X-100 (1 mL at 0.1% (v/v)) was added to the wells and they were incubated in the dark for 20 min at room temperature. Measurement of the fluorescence was immediately undertaken by a spectrofluorophotometer (RF-5001PC, Shimadzu, Kyoto, Japan) at 495 nm excitation wavelength and 525 nm emission wavelength in a 1 mL cuvette.
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