Enzyme linked immunosorbent assay kit

Mesalazine was purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd. (Shanghai, China); TNBS and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA); methanol and acetonitrile were purchased from CNW Technologies (Shanghai, China); 2-chloro-L-phenylalanine was purchased from Shanghai Hengbai Biotech Co., Ltd. (Shanghai, China); and enzyme-linked immunosorbent assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

Oxidative stress indexes such as malondialdehyde (MDA) concentration, hydrogen peroxide (H₂O₂) content, superoxide dismutase (SOD) activity, and glutathione (GSH) content, as well as levels of inflammatory factors such as myeloperoxidase (MPO), interleukin-6 (IL-6), and interleukin-1β (IL-1β), were detected using corresponding kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Concentration of 20-hydroxystilbenetetraenoic acid (20-HETE) was determined using an enzyme-linked immunosorbent assay kit (Jianglai Industrial Limited by Share Ltd., Shanghai, China). Moreover, concentrations of PGE₂, LTB₄, and phospholipase A₂ (PLA₂) were determined by corresponding enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioengineering Institute). All operations were performed strictly as described in the kit manufacturer's protocol. The catalog numbers of all kits are listed in Table 1.

At the end of the experiment, blood samples were collected from birds via the wing vein on sampling days as previously described. The serum was centrifuged at 3,000 rpm for 15 min at room temperature. Serum samples were aspirated by a pipette and stored in 1.5 ml tubes at -20°C until analyzed. The serum concentrations of total protein (TP), albumin (ALB), globulin (GLB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglycerides (TGs), calcium (Ca), phosphate (P), immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) were measured by an automatic biochemical analyzer (7600; Hitachi, Tokyo, Japan), following the manufacturer’s instructions. Also, the levels of total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), malondialdehyde (MDA), interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were evaluated with enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Biology Engineering Institute, Nanjing, China). All operations are in accordance with the instructions. Hens were humanely euthanized using an injection of pentobarbital sodium (0.4 ml kg/body weight; Sile Biological Technology Co. Ltd., Guangzhou City, China).

Approximately 10 mL of blood was collected from the jugular vein of each sheep using a sterile transfusion needle and negative-pressure blood aspiration tube. Serum was obtained via centrifugation at 3,500 r/min for 10 min and stored at −20°C. Serum hormone (growth hormone, insulin-like growth factor-1, and leptin) and immune (IgA, IgG, and IgM) concentrations were determined according to the manufacturer’s instructions using enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Biotechnology Co., Nanjing, China).

Transforming growth factor-β (TGF-β), D-lactate (D-Lac), diamine oxidase (DAO), and 8-hydroxy-2′-deoxyguanosine (8-OH-dG) in the serum were measured through enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China).

The primary mouse macrophage RAW 264.7 cell line was obtained from Prof. Zhang's lab., Lanzhou Institute of Husbandry and Pharmaceutical Sciences, CAAS (Lanzhou, China). RAW264.7 cells (500 μL) in the logarithmic growth stage were inoculated on 24-well plates at a concentration of 1 × 10⁵/mL. The medium was removed after culture at 37 °C for 24 h, and then equal amounts of medium containing four essential oils (compounds) at different concentrations were added. One hour later, 5 μL of LPS (0.5 mg/mL) was added to each well except the blank control group, and the cells were cultured for another 24 h to induce inflammation. DEX (5 μg/mL) was used as a positive control. Finally, the cells were collected to measure the levels of nitric oxide (NO), interleukin-6 (IL-6) and superoxide dismutase (SOD) using enzyme-linked immunosorbent assay kits from Nanjingjiancheng Bio (NJJCBIO, China), and the absorbance was measured using a Multiskan Go Microplate Spectrophotometer (Thermo Scientific., U.S.A).