A piece of testicular tissue from each mouse in each of the four groups was immediately homogenized in ice-cold PBS. Total protein extraction and protein concentration measurements were performed as described above to analyze the enzyme activities. The T-AOC of testicular tissue was measured by the rapid 3-ethylbenzthiazoline-6-sulfonic acid method (cat# S0121, Beyotime). Total SOD activity and GPx activity in testicular tissue were measured with a total superoxide dismutase assay kit (cat# S0109, Beyotime) with nitroblue tetrazolium (NBT) and a glutathione peroxidase assay kit (cat# S0056, Beyotime) with nicotinamide adenine dinucleotide phosphate (NADPH), respectively. The levels of hydrogen peroxide and MDA in testicular tissue were detected using a hydrogen peroxide detection kit (cat# S0038, Beyotime) and an MDA detection kit (cat# S0131, Beyotime) based on the chromogenic reaction between MDA and thiobarbituric acid (TBA).23 (link)24 (link)25 (link)26 (link) The T-AOC, SOD, GPx, hydrogen peroxide, and MDA levels were normalized to that of the total protein. Five or six mice from each group were used in these experiments to measure the level of oxidative stress in testicular tissue.
Hydrogen peroxide detection kit
Manufactured by Beyotime
Sourced in China
The Hydrogen Peroxide Detection Kit is a laboratory tool used to detect the presence and concentration of hydrogen peroxide in various samples. It provides a quantitative analysis of hydrogen peroxide levels.
Automatically generated - may contain errors
Lab products found in correlation
Mda detection kit, by Beyotime (1 mentions)
Glutathione peroxidase assay kit, by Beyotime (1 mentions)
Total superoxide dismutase assay kit, by Beyotime (1 mentions)
5 mthf, by Yuanye Bio-Technology (1 mentions)
Epigallocatechin gallate (egcg), by Solarbio (1 mentions)
L ascorbic acid, by Maclin Biochemical Technology (1 mentions)
4 protocols using hydrogen peroxide detection kit
1
Testicular Oxidative Stress Analysis
2
Visualizing Tomato Leaf H2O2 and Immune Genes
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Two hours after rutin spraying, 3,3-diaminobenzidine (DAB) staining was used to detect the the accumulation of H2O2 in tomato leaves, and RNA was extracted to detect the expression of early immune-related genes (RBOHD, MAPK3 and MAPK6). Tomato leaves were immersed in a solution of DAB (1 mg/mL), subjected to vacuum infiltration for 30 min, and then washed three times. The leaves were exposed to light illumination at 28 °C for 8 h to allow H2O2 to react. Chlorophyll was removed by immersing the leaves in boiling ethanol (95%) for 10 min. The concentration of H2O2 was determined using a hydrogen peroxide detection kit (Beyotime, Shanghai, China) following the manufacturer's instructions.
3
EGCG and 5-MTHF Antioxidant Evaluation
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EGCG (≥98 %) and 10 × phosphate buffer were purchased from Solarbio (Beijing, China), while L-ascorbic acid (>99 %) was purchased from Macklin (Shanghai, China). 5-MTHF (≥98 %) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China), and 5-MTHF-13C5 (99 atom % 13C, 95 % (CP)) was purchased from Sigma-Aldrich (Shanghai) Trading Co. Ltd (Shanghai, China). Hydrogen peroxide detection kit was purchased from Beyotime (Shanghai, China). EGCG was dissolved in methanol and diluted with PBS to prepare a 50 mM stock solution. L-ascorbic acid was dissolved in PBS to create an 11 % stock solution. The handling protocols for 5-MTHF and 5-MTHF-13C5 solutions adhered to the methodologies detailed in Ref. [26 (link)].
4
Extraction and Quantification of Oxidative Stress Markers
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The extraction of hydrogen peroxide (H2O2), superoxide anion (O2−) and malondialdehyde (MDA) were carried out as described by Wang et al. [20 (link)]. Weigh 0.5 g leaf or 1.0 g root fresh sample, grind the fresh sample into homogenate on ice with extraction solution, the supernatant was extracted by centrifugation for the determination of above parameters and antioxidant enzyme activity.
The H2O2 content was measured by hydrogen peroxide detection kit (Beyotime Institute of Biotechnology, Shanghai, China).
The O2− release rate was assayed by hydroxylamine method [45 (link)]. After mixed reaction system, the mixture was kept at 25 °C for 20 min and the OD value was measured under OD530 nm of spectrophotometer.
The MDA content was determined according to Tan et al. [49 (link)]. In total, 4 mL TCA-TBA mixture solution was added with 2 mL extraction, boiling water bath for 20 min, centrifugation at 4000× g for 10 min, supernatant was taken and read at OD450 nm, OD532 nm and OD600 nm under a visible light spectrophotometer.
The H2O2 content was measured by hydrogen peroxide detection kit (Beyotime Institute of Biotechnology, Shanghai, China).
The O2− release rate was assayed by hydroxylamine method [45 (link)]. After mixed reaction system, the mixture was kept at 25 °C for 20 min and the OD value was measured under OD530 nm of spectrophotometer.
The MDA content was determined according to Tan et al. [49 (link)]. In total, 4 mL TCA-TBA mixture solution was added with 2 mL extraction, boiling water bath for 20 min, centrifugation at 4000× g for 10 min, supernatant was taken and read at OD450 nm, OD532 nm and OD600 nm under a visible light spectrophotometer.
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