The intracellular levels of GSH were measured by GSH Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The OD value of GSH was detected with a fluorescence microplate reader (Tecan, Männedorf, Switzerland) at 420 nm. In addition, the intracellular levels of O2− were detected by Superoxide Assay kit (Beyotime Institute of Biotechnology), and the fluorescence value of O2− was measured at a wavelength of 550 nm.
Superoxide assay kit
Manufactured by Beyotime
Sourced in China
The Superoxide assay kit is a laboratory product designed to detect and quantify superoxide (O2−) levels in various samples. It provides a reliable and efficient method for measuring this important reactive oxygen species (ROS) using a colorimetric or fluorometric approach. The kit offers a straightforward protocol and reagents necessary to conduct the analysis.
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Lab products found in correlation
Microplate reader, by Agilent Technologies (2 mentions)
Ros assay kit, by Beyotime (2 mentions)
Microplate reader, by Tecan (2 mentions)
Fluorescence microplate reader, by Tecan (1 mentions)
Gsh assay kit, by Beyotime (1 mentions)
12 well culture plate, by Corning (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Apocynin, by Merck Group (1 mentions)
96 well dishes, by Corning (1 mentions)
Dihydroethidium, by Thermo Fisher Scientific (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Bca assay kit, by Transgene (1 mentions)
Dcfh da, by Beyotime (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Lipid peroxidation mda assay kit, by Beyotime (1 mentions)
Ldh cytotoxicity assay kit, by Beyotime (1 mentions)
96 well plate, by Agilent Technologies (1 mentions)
Rhodamine 123 staining, by Beyotime (1 mentions)
Xfe96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
27 protocols using superoxide assay kit
1
Intracellular GSH and O2- Quantification
2
Superoxide Measurement in Leptospira-Infected Cells
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Superoxide was measured using a superoxide assay kit (Beyotime, China) as previously described [26 (link)]. Briefly, THP-1 or J774A.1 cells (1 x 105 per well) were seeded into 12-well culture plates (Corning, USA) for incubation overnight at 37°C. The cell monolayers were washed thoroughly with PBS and then infected with harvested L. interrogans strain Lai at a MOI of 100 for co-incubation at 37°C for 2, 4, 12 or 24 h as described above. Because superoxide can deoxidize WST-1 and produce a soluble orange formazan [26 (link), 27 (link)], the optical density of each microplate was measured at 450 nm using a Bio-RAD microplate reader (Bio-Rad, Hercules, CA). For inhibitory tests, cells were pretreated with apocynin (100 μmol/L) (Sigma-Aldrich, St. Louis, Mo.) to inhibit NADPH oxidase activity at a final concentration of 0.5 μmol/L for 1 h at 37°C, and the subsequent experimental steps were the same as described above.
3
Quantifying Superoxide Levels
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Superoxide levels were assessed using a Superoxide Assay Kit (S0060, Beyotime, China) following the manufacturer's instructions. Briefly, the working solution for each detection was prepared in the following proportions: 200 μl superoxide detection buffer, 10 μl WST-1 solution, and 2 μl catalase. A volume of 200 μl superoxide test solution was added to each well and incubated at 37°C for 3 minutes. Absorbance was measured at a wavelength of 450 nm using a microplate reader.
4
Superoxide Assay for JS-K Treatment
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Intracellular superoxide levels were measured with a Superoxide Assay Kit (Beyotime) according to the manufacturer’s protocol. Briefly, cells were seeded into 96-well dishes (Corning) and treated with JS-K (1 μM, 2 μM and 5 μM) for 6 h. The culture medium containing JS-K was replaced with fresh medium, and the cells were washed with PBS. Superoxide detection reagent was added into each well (200 μL/well) and incubated at 37 °C for 20 min. The absorbance was recorded at 450 nm in a 96-well plate reader (EnSpire 2300 Multilabel Reader, PE).
5
Superoxide Anion Detection Assay
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Dihydroethidium (Life Technologies) was added to each well at a concentration of 10 μM. After 30 min, the samples were analysed with a flow cytometer (20,000 events) or fluorescence microplate (Equal protein lysate) to detect the fluorescence. A Superoxide Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China) was also used to detect superoxide anionin accordance with the manufacturer’s instructions.
6
Intracellular Oxidative Stress Quantification
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The intracellular superoxide was quantitatively measured with the Superoxide Assay Kit (S0060, Beyotime, China) to evaluate the intracellular oxidative stress level after culturing for 3 and 6 h. The culture medium was collected and diluted to a proper concentration before the assay kit was added. After cultivation for another 3 min at 37 °C, OD450 and OD600 were monitored to quantitatively determine the superoxide.
Hydrogen peroxide level was measured in cell lysates using a colorimetric assay kit according to the manufacturer instructions (Sigma Aldrich, USA, cat. No. MAK311).
Hydrogen peroxide level was measured in cell lysates using a colorimetric assay kit according to the manufacturer instructions (Sigma Aldrich, USA, cat. No. MAK311).
7
Evaluating Antioxidant Effects on ADSCs
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To further determine the antioxidative effect of GSH and melatonin on ADSCs, different passages of ADSCs were collected, and the content of NOXs was measured using a NOXs test Kit (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) following the manufacture’s protocol. Finally, the content of NOXs was normalized to the corresponding protein concentration of ADSCs, which was analyzed by a bicinchoninic acid (BCA) assay kit (TransGen Biotech, Beijing, China). To further determine the content of superoxide in ADSCs, a Superoxide Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China) was used. Briefly, the ADSCs were plated at a density of 5 × 103 cells/well in 96-well plates. Twenty-four hours later, the cell culture supernatant was discarded and the cells were washed with 0.1 M PBS for three times; then, 200 μL superoxide detection solution was added into each well at room temperature for 10 min; finally, the absorbance of each well was measured using a microplate reader (Spectra Max M5; Molecular Devices USA) at 450 nm.
8
Quantifying Neuronal Superoxide Levels
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The cellular superoxide level was examined using a superoxide assay kit (Beyotime, Wuxi, China) according to the manufacturer’s instructions. Briefly, neuronal cells were cultured onto six-well plates at a density of 3 × 105 cells/well. Following the indicated treatment, the superoxide detection reagent (100 μL/well) was added for 20 min at room temperature. The absorbance, reflecting the superoxide assay, was recorded at 450 nm.
9
Superoxide Assay for TGF-β2 Impact
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Superoxide levels were estimated using a superoxide assay kit (#S0060, Beyotime, Shanghai, China). Cells were seeded in 96-well plates at a density of 5 × 104 cells/ml. After the cells were fully adherent, 200 µl of superoxide assay solution with or without TGF-β2 was added to each well. The OD was measured at 450 nm after 1 h using a microplate reader (BioTek, Vermont, USA).
10
Evaluating ROS and Oxidative Stress
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The ROS production
was evaluated by using DCFH-DA, a nonfluorescent compound, which can
readily diffuse into water and interact with ROS. After incubation
with different concentrations of BQAS for 3 h, the bacterial suspensions
were washed and stained with 10 μm DCFH-DA (Beyotime Biotechnology)
for 20 min in dark at 25 °C. Green fluorescent ROS-producing
cells were visualized with a fluorescence microscope at a 488 nm excitation
wavelength. The areas of observation were randomly photographed with
a Nikon fluorescence microscope.
The possibility of superoxide
anion (O2•–) production was evaluated
to determine the oxidative stress using the superoxide assay kit (Beyotime
Biotechnology). Briefly, 200 μL of the bacterial suspension
was centrifuged and washed twice with sterile water, followed by adding
200 μL of detection solution and incubating at 37 °C for
5–10 min. Next, the bacterial samples were incubated with different
concentrations of BQAS under the same conditions as described above.
Finally, the samples were examined by a microplate absorbance reader
at 450 nm.
was evaluated by using DCFH-DA, a nonfluorescent compound, which can
readily diffuse into water and interact with ROS. After incubation
with different concentrations of BQAS for 3 h, the bacterial suspensions
were washed and stained with 10 μm DCFH-DA (Beyotime Biotechnology)
for 20 min in dark at 25 °C. Green fluorescent ROS-producing
cells were visualized with a fluorescence microscope at a 488 nm excitation
wavelength. The areas of observation were randomly photographed with
a Nikon fluorescence microscope.
The possibility of superoxide
anion (O2•–) production was evaluated
to determine the oxidative stress using the superoxide assay kit (Beyotime
Biotechnology). Briefly, 200 μL of the bacterial suspension
was centrifuged and washed twice with sterile water, followed by adding
200 μL of detection solution and incubating at 37 °C for
5–10 min. Next, the bacterial samples were incubated with different
concentrations of BQAS under the same conditions as described above.
Finally, the samples were examined by a microplate absorbance reader
at 450 nm.
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