Tnf α enzyme linked immunosorbent assay elisa kit

Blood samples were stored for 6 h at 4°C, then centrifuged for 20 min at 3,000 rpm/min and 4°C. The supernatant was collected and stored at -80°C until TNF-α concentration was measured.
Fat on the outer HG wall was removed and the tissue was cut open on a sterile bench and the contents removed. The tissue was washed for 30 s in phosphate-buffered saline (PBS) at 4°C to remove mucus, then cut into pieces and weighed. The pieces were transferred to a Tissuelyser-24, and nine times the volume of PBS buffer was added before homogenization for 30 s at 4°C. The homogenate was centrifuged for 20 min at 3,000 rpm and 4°C. The supernatant was collected and stored at -80°C until measurement of TNF-α concentration.
A TNF-α enzyme-linked immunosorbent assay (ELISA) kit (Cusabio Biotech, Wuhan, China) was used to measure TNF-α concentration according to the manufacturer’s instructions. Absorbance was read on a full-wavelength Multiskan GO microplate reader (Thermo Fisher Scientific, Waltham, MA, United States) with a sensitivity <50 pg/mL.

The following reagents were used in the study: Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Wisent (Montreal, QC, Canada). Puromycin (PM), penicillin, and streptomycin were purchased from Sigma (St. Louis, MO, USA). SDS-polyacrylamide gel electrophoresis and 10% buffered neutral formaldehyde were purchased from Beyotime (Shanghai, China). Nitrocellulose membranes were provided by Millipore (Billerica, MA, USA). FITC-labeled anti-mouse F4/80, PE-conjugated anti-mouse programmed death ligand 1 (PD-L1), and PE-Cy5-labeled anti-mouse CD80 monoclonal antibodies were obtained from eBioscience (San Diego CA, USA) for flow cytometry analysis. Antibodies against MMP-2, MMP-9, and GAPDH used for Western blotting were manufactured by Proteintech (Chicago, IL, USA). Rat anti-mouse F4/80, CD68, IL-10, TGF-β, VEGF, and anti-mouse major histocompatibility complex class II (MHCII) monoclonal antibodies that recognize cells from both BALB/c and C57 BL/6 mice were manufactured by Abcam (Cambridge, MA, USA) for immunohistochemistry assays. The TNF-α enzyme-linked immunosorbent assay (ELISA) kit was provided by CUSABIO (Wuhan, China). The interleukin-6 (IL-6) ELISA kit was obtained from RayBiotech (Norcross GA, USA). The Greiss Reagent System determined for nitrite was purchased from Promega Biotech Company (Madison, WI, USA).