Bc0025

Homozygous T₃ transgenic wheat lines were selected for the assays of growth (root length and seedling height) and physiological analysis. Samples were collected at 0 h (control), 5 h (T1 stage), and 24 h (T2 stage) after salt stress, and at 1 h (R stage) after recovery. The assay kits were applied for measuring proline content (Cas no.: BC0295, Solarbio, Beijing, China), soluble sugar (Cas no. BC0035, Solarbio), and MDA (Cas no.: BC0025, Solarbio) in accordance with the manufacturer’s instructions. Three biological replications were performed, and each replication contained at least 10 seedlings for the phenotype and physiological determination in this experiment.

The measurement of antioxidant capacity in rumen epithelial tissue was followed our previous protocol (53 (link)). Briefly, 0.1 g of rumen epithelial tissue was immediately extracted and homogenized with 1 mL of PBS buffer (0.01 mol/L; pH 7.2 to 7.4) on ice, and the homogenized tissue was then centrifuged at 13,400 × g at 4°C for 10 min to collect the supernatant for subsequent analysis. The protein concentration was measured using a total protein quantitative assay kit (A045-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The T-AOC (BC1315; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), the activity of GSH-Px (BC1195; Solarbio), and the MDA content (BC0025; Solarbio) were assayed using biochemical kits according to the manufacturers’ protocols.

Following the LSS experiment, EA.hy926 cells were collected to measure secreted superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, glutathione (GSH) content and glutathione disulfide (GSSG) content using ELISAs (S8530, BC0025, BC1170, BC1185, Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturers’ protocols.
Following the LSS experiment, EA.hy926 cells were also incubated with the ROS-specific fluorescent dye dihydroethidium (DHE S0063, 50 μM, Beyotime Institute of Biotechnology). Following the incubation, the EA.hy926 cells were washed in PBS twice and then visualized and analyzed via fluorescence microscopy. The fluorescence intensity was semi-quantified from ≥3 random fields of view per slide from three different slides.

The hippocampi were harvested to determine SOD and MDA levels. The hippocampus was weighed, extracting solutions from the SOD assay kit (BC0025, Solarbio, Beijing, China) or MDA assay kit (BC0175, Solarbio) were added, and the samples were homogenized on ice. The supernatants were placed on ice and analyzed per the manufacturer's instructions. The absorbance of each sample was measured at the indicated wavelengths (SOD: 560 nm; MDA: 600 nm, 532 nm, and 450 nm), and SOD and MDA levels were calculated based on a prepared standard curve.

Commercial kits were used to measure SOD activity (BC0175, Solarbio, China), as well as concentration of MDA (BC0025, Solarbio, China), GSH (BC1175, Solarbio, China), and ATP (BC0305, Solarbio, China). Briefly, fresh hippocampal samples were incubated in extraction solutions and completely homogenized on ice. Following centrifugation at 4°C for 10 minutes, the supernatants were collected to perform follow-up tests according to the manufacturer's instructions.

Dihydroethidium (DHE) staining was used to evaluate intracellular ROS levels in post‐TBI brains. Briefly, each brain was cut into 15‐μm‐thick sections, which were then stained using DHE (Yesen, 50102ES02) for 30 minutes and imaged using a laser scanning confocal microscope (A1 Si, Nikon). Assays for determining malondialdehyde (MDA) levels (BC0025, Solarbio) and manganese superoxide dismutase (MnSOD) activity (JL20470, Jiang Lai) were performed using commercial assay kits in strict accordance with the manufacturer's protocols.