Mda assay kit

After intravenous injection of Fe₃O₄ NPs (100 mg/kg) for 6 h, the mice were sacrificed and the serum, heart, and liver were collected. The harvested heart and liver were washed with cold saline and homogenized. The serum and homogenates were determined using MDA assay kits (Nanjing Jiancheng Bioengineering Institute). For GSH pretreatment group, the GSH was orally administrated at a dosage of 800 mg/kg. 2 h later, the Fe₃O₄ NPs were injected.

Malondialdehyde (MDA) assay was performed to measure lipid peroxidation. The level of superoxide dismutase (SOD) activity indirectly reflects the ability of cells to remove ROS. The hippocampal tissues were isolated from the brain, homogenized with lysis buffer, and centrifuged to get the supernatant. The MDA and SOD activity levels were detected using commercially available MDA assay kits (Jiancheng Biotech, Nanjing, China) and SOD assay kits (Jiancheng Biotech, Nanjing, China) according to the manufacturer’s protocols.

The activities of superoxide dismutase (SOD), glutathione S-transferase (GST) and glutathione peroxidase (GPX), concentrations of glutathione (GSH) and malondialdehyde (MDA) in liver and serum were measured using SOD, GST, GPX, GSH and MDA assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacture’s protocols, respectively.

Total 50 mg of kidney from sham- or CLP-operated mice (n = 5 per group) were placed in normal saline and homogenized on ice. The homogenate was absorbed (3,000 rpm, 10 min, 4°C) to further analyze. The clarified supernatants were dilute 50 times. Content of protein was measure using a Bicinchoninic Acid (Thermo Fisher, United States). The levels of malondialdehyde (MDA) were determined with MDA assay kits (Nanjing Jiancheng). Total 40 mg of kidney tissue from sham- or CLP-operated mice (n = 5 per group) were placed in fresh lysis buffer, homogenized on ice and then were centrifuged at 10,000 rpm for 5 min to detect the content of IL-6 and TNF-α(Cloud-Clone Crop, Wuhan, China). Blood samples (n = 5 per group) were obtained from the eyeballs of the mice in each group, stratified, and centrifuged (3,000 rpm, 15 min). Supernatant were aspirated and saved in a centrifuge tube to measure the level of S100A9 (Cloud-Clone Crop, Wuhan, China).

The APS (>80%) used in the mice was provided by Nanjing Jingzhu Biotechnology Co. Ltd. (Nanjing, China). The APS (>95%) used in cell experiments was purchased from PharmaGenesis, Inc. (America). Tunicamycin (TM) was purchased from Sigma (America). T-AOC, GSH, SOD, and MDA assay kits were obtained from Jiancheng Biotechnology (Nanjing, China). The total protein assay kit was purchased from the Biyuntian Company (Nanjing, China).

Treated cells and supernatants of cells were collected at different intervals (2, 4, 6, and 8 h) after blast simulation. We evaluated oxidative stress using the protocols of SOD and the MDA assay kits (Jiancheng Bioengineering Ltd., Nanjing, China). Protein content was measured according to the manufacturer’s instructions.