Il 18 elisa kit

Abdominal aorta and portal venous blood were centrifuged at 3000 RPM for 10 min at 4°C, and the upper serum was collected. To obtain tissue homogenates, 100 mg of liver tissue was mixed with 0.9 mL of isopropanol, followed by centrifugation at 3000 RPM for 10 min at 4°C, and subsequently the tissue homogenate supernatant was collected. Abdominal aorta serum TC, TG, LDL, and high-density lipoprotein (HDL) levels, as well as liver TC and liver TG levels, were detected by using an automatic biochemical analyzer (Hitachi, Japan). Portal venous serum LPS levels were measured using a Quantitative Chromogenic Tachypleus Amebocyte Lysate (TAL) For Endotoxin Detection Kit (Chinese Horseshoe Crab Reagent Manufactory, CO., Ltd., China) according to the manufacturer's instructions. Abdominal aorta serum IL-1β and IL-18 levels were tested using enzyme-linked immunosorbent assay (ELISA) kits (the IL-1β ELISA kit was obtained from eBioscience, USA. The IL-18 ELISA kit was obtained from Invitrogen Corporation, USA).

Urinary NGAL, Kim-1 and IL-18 levels were analyzed using commercially available enzyme-linked immune absorbent assay (ELISA) kits: human NGAL ELISA kit (BioPorto Diagnostics, KIT036), human kidney injury molecule 1 (Kim-1) ELISA kit (Cusabio Biotech, CSB-E08807h) and human Interleukin-18 (IL-18) ELISA Kit (Invitrogen, KHC0181). All procedures were performed according to the manufacturer’s instructions.

The 200–300 mg of colon tissue was washed in cold PBS supplemented with penicillin and streptomycin. These segments were cut into small pieces and cultured in 12-well flat bottom culture plates (Falcon) in serum-free RPMI medium. 100 U/mL penicillin G and 100 μg/mL streptomycin sulfate (Thermo Fisher Scientific) was supplemented to prevent bacteria growth. After incubation at 37 °C for 24 h, the medium was collected and examined for cytokine and chemokine production with MCP-1 ELISA kit, MIP-2 ELISA kit, IL-6 ELISA kit, TNF-α ELISA kit, KC ELISA kit, and IFN-γ ELISA kit, CCL2 ELISA kit, CXCL2 ELISA kit, CXCL8 ELISA kit, and IL-18 ELISA kit according to the manufacturer’s instruction. CXCL8 ELISA kit and IL-18 ELISA kit were purchased from Thermo Fisher Scientific, other ELISA kits were purchased from NeoBioscience. Cytokine and chemokine production is normalized by total colon tissue weight (whole-colon culture).
IL-37 ELISA kit (R&D Systems) was used to detect the levels of IL-37 in the colon tissue homogenates and human serum. IL-37 production in colon tissue homogenates is normalized by total protein amount (crypt protein lysate) measured by BCA analysis (Pierce). IFN-γ ELISA kit (BioLegend) was used to detect the levels of IFN-γ in cell culture supernatants. All assays were performed according to the manufacturers’ instructions.

Supernatants from cell cultures were collected, filtered by 0.22 μm Hydrophilic low protein binding Durapore membrane (Merck Millipore, Darmstadt, Germany) and stored at -80°C. Cytokine determinations were performed using the Bio-Plex 200 system with a multiplex kit #M60009RDPD (BioRad Laboratories, Hercules, CA, USA). Concentrations of IL-18, IL-1β, and IFN-β were measured by following the manufacturer’s protocols for the IL-18 ELISA kit (# KHC0181; Thermo Fisher Scientific, Waltham, MA), OptEIA mouse IL-1β ELISA kit (#559603;BD Biosciences, Franklin Lakes, NJ), and VeriKine Mouse IFN-b ELISA Kit #42400 PBL Assay Science, Piscataway, NJ, respectively.

The BM plasma samples were collected from EDTA stabilized bone marrow of 70 ND AML patients and 15 controls, and supernatants of primary leukemia cells or THP-1 cells were collected and stored at -80°C for determination of cytokines. Human IL-1β ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA) and IL-18 ELISA kit was purchased from eBioscience (San Diego, CA). ELISA was performed in accordance with the manufacturer’s instructions.