ROS generation in cells was measured using ESR according to a previous study [19 (link)]. Cells were seeded on a glass cover slip (49 × 5 × 0.2 mm) and incubated until confluence. Then, cells were exposed to 10 μM cisplatin for 1 h. After treatment, cells were suspended in a respiration solution containing 5 mM succinate (Sigma-Aldrich Japan K.K., Tokyo, Japan), 5 mM glutamate (Sigma-Aldrich Japan K.K., Tokyo, Japan), 5 mM malate (Sigma-Aldrich Japan K.K., Tokyo, Japan), 5 mM NADH (Dojindo, Kumamoto, Japan), and a spin trapping agent (5.9% (v/v) DMPO (Labotec Co., Tokyo, Japan)). ESR spectra were recorded using a JEOL-TE X-band spectrometer (JEOL, Tokyo, Japan). All ESR spectra were obtained under the following conditions: 7.5 mT sweep width, 0.1 mT modulation width, 0.1 s time contrast, 335.5 mT center field, and 9.4 GHz frequency.
Te x band spectrometer
Manufactured by JEOL
Sourced in Japan
The JEOL-TE X-band spectrometer is a laboratory instrument designed for electron paramagnetic resonance (EPR) spectroscopy. It operates at X-band microwave frequencies and is used to detect and analyze the properties of paramagnetic species, such as free radicals, transition metal ions, and some organic compounds.
Automatically generated - may contain errors
Lab products found in correlation
Glutamate, by Merck Group (5 mentions)
Succinate, by Merck Group (5 mentions)
Nicotinamide adenine dinucleotide nadh, by Merck Group (3 mentions)
Malate, by Fujifilm (2 mentions)
5 5 dimethyl 1 pyrroline n oxide dmpo, by Dojindo Laboratories (2 mentions)
Nicotinamide adenine dinucleotide, by Merck Group (2 mentions)
Malate, by Merck Group (1 mentions)
5 5 dimethyl 1 pyrroline n oxide, by Merck Group (1 mentions)
14 protocols using te x band spectrometer
1
ROS Generation Measurement in Cells
2
Measuring ROS Generation in Cells by ESR
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ROS generation in cells was measured by electron spin resonance (ESR). The 4T1E cells were seeded on a glass cover slide (49 mm × 5 mm × 0.2 mm) and incubated until they were confluent. The cells were incubated at 37 °C or 42 °C for 1 h. The cover slide, with cells attached, was placed in a glass holder and immersed in a respiratory buffer containing 5 mM succinate, 5 mM malate, 5 mM glutamate, 5 mM nicotinamide adenine dinucleotide, and 10 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; Dojindo, Tokyo, Japan). All ESR spectra were obtained using a JEOL-TE X Band spectrometer (JEOL Ltd., Tokyo, Japan) under the following conditions: 7.5 mT sweep width, 1000 gain, 0.1 mT modulation width, 0.1 s time contrast, 335.5 mT center field, 9.4 GHz frequency, and 10 mW incident microwave power.
3
Quantifying Intracellular ROS Levels
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Intracellular ROS production levels in HT-treated MCF-7 and MDA-MB-453 cells were measured using electron spin resonance (ESR) in accordance with the protocol described in a previous report [22 (link)]. The cells were seeded on cover slides (49 × 5 × 0.2 mm) and incubated overnight. The cells were incubated at 37 °C or treated at 42 °C for 1 h, and the slides were then immersed in a respiratory solution containing 5 mM succinate (Sigma-Aldrich Co., St. Louis, MO, USA), glutamate (Sigma-Aldrich Co.), malate (Wako Pure Chemical Industries, Ltd.), nicotinamide adenine dinucleotide (NADH) (Sigma-Aldrich Co.), and a 5 μL 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) solution (DOJINDO LABORATORIES). The cell-attached glass cover slide was placed on a tissue glass and inserted into the ESR apparatus. All ESR spectra were obtained using a JEOL-TE Xband spectrometer (JEOL Ltd., Tokyo, Japan) under the following conditions: 7.5 mT sweep width, 1000 gain, 0.1 mT modulation width, and 10 mW incident microwave power. ESR spectra data were analyzed using a Win-Rad Radical Analyzer System (Radical Research Co., Ltd., Tokyo, Japan).
4
Intracellular ROS Generation Measured by ESR
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Intracellular ROS generation after iron treatment was detected by electron spin resonance (ESR) according to a previous study.(24 (link)) Briefly, cells were seeded on a sterilized glass cover slide (49 × 5 × 0.2 mm) at confluency and incubated overnight. Cells were then exposed to fresh medium containing 500 µM FeSO4 for 1 h and then immersed in the respiratory buffer containing 5 mM succinate (Sigma-Aldrich Japan K.K., Tokyo, Japan), 5 mM malate (Wako), 5 mM glutamate (Sigma-Aldrich Japan K.K.), 5 mM nicotinamide adenine dinucleotide (NADH) (Sigma-Aldrich Japan K.K.) and 5 µl 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (Dojindo Laboratories, Kumamoto, Japan). The slide was set in a tissue glass, and ESR spectra were obtained using a JEOL-TE X-band spectrometer (JEOL, Ltd., Tokyo, Japan) using the following measurement conditions: 10 mW incident microwave power, 9.4 GHz frequency, and 0.1 mT field modulation amplitude. The experiments were performed three times independently.
5
Mitochondrial Radical Detection by ESR
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Isolated mitochondria were prepared from RGM1 cells with MITOISO2, according to the manufacture’s instruction and as reported previously.(1 (link),4 (link)) The mitochondrial pellet was suspended and treated with 1 mM IND/2 mM aspirin for 1 h after pretreatment with 5-µg/ml Qing Dai for 1 h. After incubation, the mitochondrial pellet was suspended with a respiratory solution (5 mM succinate, 5 mM glutamate, 5 mM malate, and 5 mM NADH) containing a spin-trapping agent, either 10 mM CYPMPO or 20 mM DMPO. The solution was immediately transferred to a quartz flat cell (60 × 6 × 0.3 mm; RDC-60, Radical Research). The protein concentration in the final reaction mixture was 250 µg/ml, as evaluated using the above-mentioned method (Bio-Rad Laboratories, Hercules, CA). The electron spin resonance (ESR) spectra were recorded using a JEOL-TE X-band spectrometer (JEOL, Tokyo, Japan). All ESR spectra were obtained under the following conditions: 10-mW incident microwave power, 100-kHz modulation frequency, 0.1-mT field modulation amplitude, and 15-mT scan range. Analysis of the hyperfine splitting constants and spectral computer simulation were performed using a Win-Rad Radical Analyzer System (Radical Research). All ESR spectra shown are representative of at least three independent experiments.
6
ROS Quantification via ESR Spectroscopy
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ROS generation in cells was measured using electron spin resonance (ESR) according to previous study.(13 (link)) Cells were seeded on a glass cover slide (49 × 5 × 0.2 mm) and incubated overnight. Cells were exposed to the medium containing 1 mM IND for 1 h. Cells were immersed in respiratory buffer containing 5 mM succinate (Sigma-Aldrich Japan K.K., Tokyo, Japan), 5 mM malate (Wako Pure Chem. Ind., Ltd., Osaka, Japan), 5 mM glutamate (Sigma-Aldrich Japan K.K.), 5 mM nicotinamide adenine dinucleotide (NADH) (Sigma-Aldrich Japan K.K.), and 10 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (DOJINDO). The cell-attached glass cover slide was placed on a tissue glass, and the ESR spectra were obtained by inserting the tissue glass into the device. All ESR spectra were obtained using a JEOL-TE X-band spectrometer (JEOL Ltd., Tokyo, Japan) under the following conditions: 20 mW incident microwave power, 9.42 GHz frequency, and 0.1 mT field modulation amplitude.
7
ESR Analysis of Laminaran's ROS Scavenging
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The ROS-scavenging ability of laminaran was measured via ESR. Laminaran was dissolved in Milli-Q water at a concentration of 500 mg mL−1. ROS levels were measured according to the method of Oowada et al. [19 (link)]; 1O2 was generated in combination with acid red and light. This reaction mixture comprised Milli-Q water with 200 µM acid red and 10 mM TEMPOL with or without 50 mg mL−1 laminaran. •OH was generated in combination with H2O2 and light. This reaction mixture comprised Milli-Q water with 10 mM H2O2 and 10 mM CYPMPO with or without 50 mg mL−1 laminaran. O2•− was generated from a xanthine/xanthine oxidase reaction. The reaction mixture comprised Milli-Q water with 20 mM hypoxanthine, 20 units mL−1 xanthine oxidase, and 10 mM CYPMPO with or without 50 mg mL−1 laminaran. The ESR spectra of the laminaran-containing mixtures were recorded using a JEOL-TE X-band spectrometer (JEOL, Tokyo, Japan) and compared with those of each mixture without laminaran. ESR spectra were obtained under the following conditions: 20 mW incident microwave power, 9.2 GHz frequency, 0.2 mT modulation width, 7.5 mT sweep width, 0.1 s time contrast, and 335.5 mT center field. All experiments were repeated three times.
8
Intracellular ROS Measurement by ESR
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Intracellular ROS was measured by ESR using living cells, as previously reported.(14 (link)) Cells were cultured on slide glass until confluent. The slide glass was immersed in acetic acid-containing medium (5 µM) for 60 min in a 5% CO2 incubator at 37°C. After incubation, the slide glass was placed on the tissue glass (Radical Research Inc., Tokyo, Japan). Eighty microliters of the solution for ESR measurement, which was prepared by dissolving respiratory substrates (5 mM succinic acid, 5 mM malic acid, 5 mM d -glutamic acid, and 5 mM NADH) and 5.9% v/v DMPO, was poured onto the tissue glass. The ESR spectra were then recorded using a JEOL-TE X-band spectrometer (JEOL, Tokyo, Japan). All ESR spectra were obtained under the following conditions: 10 mW incident microwave power, 0.1 mT modulation width, 8 min sweep time, 7.5 mT sweep width, 0.1 s time contrast, 333.5 mT center field, and 15 mT scan range. Spectral computer simulations were performed using a Win-Rad Radical Analyzer System (Radical Research).
9
Intracellular ROS Measurement by ESR
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Intracellular ROS production was measured with electron spin resonance (ESR), as described previously.(11 (link)) Cells were seeded onto glass cover slides (49 mm × 5 mm × 0.2 mm) and cultured overnight. After incubation, the slides were cultured for 6 h in medium containing 0.2 mM IND. The IND concentrations in our experiments referred to previous reports using gastric mucosa cells.(7 (link)) Cells then were shifted to culture medium supplemented with respiratory buffer solution (5 mM succinate, 5 mM glutamate, and 5 mM malate), with 5 mM nicotinamide adenine dinucleotide (Sigma-Aldrich Japan K.K.), and with 10 mM 5,5-dimethyl 1-pyrroline-N-oxide (DOJINBO LABORATORIES). The ESR spectrum measurements were performed with a JEOL-TE X-band spectrometer (JEOL, Ltd., Tokyo, Japan) using the following settings: incident microwave power, 20 mW; frequency, 9.42 GHz; and field modulation amplitude, 0.1 mT.
10
Intracellular ROS Measurement by ESR
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The intracellular levels of ROS after treatment with acetic acid were estimated by the ESR method, as described in previous reports.(13 (link),14 (link)) Briefly, RGK1 cells were cultured to confluence on small slide cover glasses (49 × 5 × 0.2 mm). These cells were treated with 0, 5, and 10 μM acetic acid-containing medium for 1 h in a 5% CO2 incubator at 37°C. The cover glass was then placed on a quartz flat tissue cell (RDC-60, 60 × 6 × 0.3 mm) (Radical Research Inc., Tokyo, Japan), and 80 μl of a solution for ESR measurement composed of 5 mM each of succinic acid, malic acid, d -glutamic acid, and NADH, and 5 μl 5,5-Dimethyl-1-pyrroline N-oxide was poured onto it. The ESR spectra were measured using a JEOL-TE X-band spectrometer (Jeol, Tokyo, Japan). All ESR spectra were obtained under the following conditions: 10 mW incident microwave power, 0.1 mT modulation width, 8 min sweep time, 7.5 mT sweep width, 0.1 s time contrast, 335.5 mT center field, and 15 mT scan range. ESR measurements and analyses were performed using a Win-Rad Radical Analyzer System (Radical Research).
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