Dy479

Murine derived hypothalamic neuronal cell lines hypoE-46 and hypoA2/12 (CELLutions Biosystems Inc. Canada) were grown and maintained in DMEM supplemented with 10 % heat-inactivated fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C under 5.0 % CO₂. Cells were grown in monolayers to 90 % confluency. Then medium was replaced by serum-free DMEM containing penicillin and streptomycin. After 4 h, cells were exposed to LPS (1 μg/ml), TNFα (100 pg/ml), IL-6 (100 pg/ml) for 24 h, or KCl (60 mM) for 15 min. After exposure, supernatant was collected to measure levels of serotonin (BAE-5900, LDN, Nordhorn, Germany), IL-6 (DY406, Abingdon, UK), TNFα (DY410, Abingdon, UK) and MCP-1 (DY479, R&D systems, Abingdon, UK) by enzyme-immuno assay. Cells were homogenized in 40 mM Tris, 1 mM EDTA, 5 mM EGTA and 0.50 % Triton X-100. Homogenates were used to measure 5-hydroxyindoleacetic acid (5-HIAA) by ELISA (MBS261481, MyBiosource, Breda, The Netherlands) and corrected for total protein content (Pierce Bicinchoninic acid Rockford, IL, USA). Cytotoxicity was determined by measuring LDH leakage and cell viability using an XTT conversion assay after 48 h of exposure (Roche Diagnostics, Mannheim, Germany). All experiments were performed three times in quadruplicate.

To measure the concentration of IFN-α, IFN-γ, TNF-α, IL2, IL12 p40, IL23, IL27, and CCL2, tumor lysates or co-culture supernatants were used to perform ELISA assays with the kits for IFN-α (R&D Systems, 42120-1), IFN-γ (eBioscience, 88-7314-88), TNF-α (eBioscience, 88-7324-86), IL-2 (Thermo Fisher Scientific, BMS601), IL-12 p40 (abcam, ab171179), IL23 (R&D Systems, M2300), IL-27 (Thermo Fisher Scientific, BMS6024), and CCL2 (R&D Systems, DY479). The experiments were conducted in triplicates.

A total of 4 × 10⁵ ID8 cells were seeded in 6-well plate in DMED without FBS and cultured for 24 h before harvesting. ELISA on mCCL2 (DY479, R&D) and CSF-1(MMC00, R&D) were performed as instructed by manufacturer.

The distal part of the colonic tissue was homogenized similar to the proximal part. Cytokine (IL-6, IL-10, MCP-1, and TNFα) levels were measured using commercially available ELISA kits (Cat# DY406, DY417, DY479, DY410, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s manual. Cytokine levels were normalized by tissue weight and expressed as ng/g tissue.

The protein levels of IL1A, CCL2, CXCL3, and CXCL5 in the conditioned medium were detected by IL1A (DY400, R&D), CCL2 (DY479, R&D), CXCL3 (EK1364, BOSTER) and CXCL5 (EK0919, BOSTER) ELISA kits. The serum S100A14, CCL2 and CXCL5 levels in breast cancer patients and healthy controls were examined using S100A14 (QS440428, Beijing Qisong Biotechnology Co., Ltd.), CCL2 (QS40081, Beijing Qisong Biotechnology Co., Ltd.), and CXCL5 (QS41512, Beijing Qisong Biotechnology Co., LTD) ELISA test kits.