O2 assay kit

During the storage process, the fruit bodies were collected at 0, 3, 6, 9 and 12 days. The fruit bodies were ground into a fine powder in liquid nitrogen, and then the fruit body tissue (1.0 g) was homogenized with 9.0 mL of normal saline. The homogenates were centrifuged at 1000g for 10 min at 4 °C. Lipid peroxidation was assayed by estimating the MDA concentration in the supernatant using a commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The mixture containing mushroom supernatant and reagents was heated at 95 °C for 40 min. The sample was quickly cooled to room temperature and then centrifuged at 4000g for 10 min. The absorbance of the supernatant was determined at a wavelength of 532 nm.
The inhibition of superoxide anion (O₂⁻) activity was performed as described by the inhibition and produced O₂⁻ Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). In this reaction system, the amount of O₂⁻ that was inhibited by 1 g of mycelial protein at 37 °C for 40 min was related to the amount of O₂⁻ inhibited by 1 mg of vitamin C as monitored at 550 nm, and this value was taken as one unit of O₂⁻ inhibition activity.

The biochemical analyses were carried out in the fresh leaves samples. MDA determination kit (A003-1), (Nanjing Jiancheng Bioengineering Institute, NJBI) according to the manufacturer’s instructions. Briefly, the leaf tissue homogenate was centrifuged at 12000 g and 4 °C for 15 min, and the supernatants were collected. MDA and thiobarbituric acid (TBA) mixture was produced during the reaction of MDA in samples with TBA, and then this mixture was measured at 535 nm and final result of MDA was expressed as mg⁻¹ protein. H₂O₂ was extracted and its content was measured by monitoring the absorbance of the titanium-peroxide complex at 405 nm according to the method of Nanjing Jiancheng Bioengineering Institute (NJBI) determination kit. The contents of H₂O₂ were demonstrated as unit mg⁻¹ protein. The contents of hydroxyl ion (^⋅OH⁻) and superoxide anion radical (^⋅O₂⁻) in the leaves of maize seedlings were determined using the commercial OH⁻ assay kit (A018) and O^⋅−₂ assay kit (A052), respectively, obtained from Nanjing Jiancheng Bioengineering Institute, China. The ^⋅OH⁻ was expressed as units mg⁻¹ protein, and one unit was the amount required to reduce 1 M of H₂O₂ in the reaction mixture per minute at 37 °C. The ^⋅O₂⁻ were demonstrated as units g⁻¹ protein, and one unit was equivalent of the value required to inhibit superoxide anion by 1 mg of VC for 40 min at 37 °C.

The commercial kits of malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) were purchased from Beyotime. O₂^‐ assay kit was obtained from Jiancheng Bioengineering. The kidney tissues were harvested after one day of I/R surgery accomplished. The levels of MDA, O₂^‐, CAT and SOD were measured by commercial kits, respectively, following manufacture's protocol. Experiments were repeated three times.