Tnf α elisa kit

IL-6 ELISA kit (PI335, Beyotime, China) and IL-1β (ml02783) and TNF-α ELISA kit (ml001696, Mlbio, China) were used to detect. Achilles tendon tissues at the edge of the healing site were triturated and lysed, and the supernatant was collected by centrifugation (1,000 x g, 4°C). Into each 96-well plate, 100 μl of sample was added. Then, 100 μl enzyme-labeled reagent was added and incubated at 37°C for 1 h. Subsequently, 50 μl of chromogenic reagent A was added into each well and an equal volume of chromogenic reagent B was added. The mixture was shaken gently to mix the two stains evenly in the dark. After 15 min, 50 μl stop solution was added to the reaction to halt color development. It was observed that the sample color changed from blue to yellow. Subsequently, the absorbance was detected. A blank well was used for zero adjustment and the detection wavelength used was 450 nm (680, Bio-Rad, USA). A standard curve was used to determine the concentrations of IL-6, IL-1β, and TNF-α.

The supernatant and serum were collected and analyzed using a mouse IL‐1β ELISA kit (mlbio, ml301814), IL‐6 ELISA kit (mlbio, ml063160), IL‐10 ELISA kit (mlbio, ml037873), MCP‐1 ELISA kit (mlbio, ml037840), IgG ELISA kit (mlbio, ml037601), IgM ELISA kit (mlbio, ml063597), TNF‐α ELISA kit (mlbio, ml002095), C3 ELISA kit (mlbio, ml002033), and MBP ELISA kit (Jin Yibai, JEB‐12718). The absorbance of each well was measured at 450 nm.

Melianodiol (MN) was isolated from the fruits of M. azedarach by our research group. The structure of MN is shown in Fig. 1A (Xu et al., 2008 (link)). NO, IL-6, IL-10, and TNF-α ELISA kits were provided by Shanghai MLBIO Biotechnology Co. Ltd. (Shanghai, China). LPS was obtained from Sigma (Sigma Aldrich, St. Louis, MO, USA). 5-Aminosalicylic acid was provided by Shanghai Aladdin Biochemical Co. Ltd. (Shanghai, China). DSS was provided by MP Biomedicals (USA) and indomethacin by Aladdin (USA). Antibodies against β-actin, JAK2, STAT3, IKKα/β, NF-κBP65, IKBα, p-IKKα/β, p-IκBα, p-P65, iNOS, eNOS, Nrf-2, HO-1, and Keap1 were provided by Cell Signaling Technology (Danvers, MA, USA). SOD, NO, GSH, and malondialdehyde (MDA) were obtained from the Nanjing Jian-Cheng Bioengineering Institute (Jiangsu, China).