Elisa assay kit

After coagulation at room temperature for 15 min, blood samples were centrifuged at 2500 rpm for 10 min to collect their supernatants. IFN-β, IL-1β, and IL-18 levels in the serum samples were measured using ELISA assay kits (Meimian, China). Optical density (OD) values at 450 nm were measured using a RT-6100 microplate reader (Rayto, USA).

Serum immunoglobulins, i.e., IgA, IgG, and IgM, were measured using ELISA assay kits (No. MM-090502, M M-040302, and MM-040202, MeiMian, China) according to the manufacturer's instructions. In brief, the standard sample or serum diluted sample was added to the enzyme plate and incubated at 37 °C for 30 min. The HRP-conjugate reagent was added to the enzyme plate and incubated at 37 °C for 30 min after washing 5 times. Then, chromogen solutions A and B were added to the enzyme plate and incubated at 37 °C for 30 min after washing 5 times. Subsequently, the absorbance was read at 450 nm within 15 min after adding the stop solution.

Samples (100 mg) from the jejunum, ileum, and hepatic tissues were homogenized with ice-cold physiological saline (1:9, w/v) and centrifuged at 2,000 × g for 20 min at 4°C. Then the supernatants were collected to measure the levels of sIgA and cytokines, including interleukin (IL)-1β, IL-10, IL-22, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ by commercially available ELISA assay kits (Mei mian, Jiangsu, China) according to the manufacturer's instructions.

Approximately 100 mg of frozen intestinal (jejunum and ileum) and hepatic tissue samples were thawed and quickly homogenized with ice-cold physiologic saline (1:9, w/v). Then the samples were centrifuged at 2,000 × g for 20 min at 4°C. The supernatants were used for further analysis. The oxidant and antioxidant indicators, including malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and GSH peroxidase (GSH-Px), were analyzed by commercially available ELISA assay kits (Mei mian, Jiangsu, China) according to the manufacturer's instructions with a Multiscan Spectrum Spectrophotometer (Tecan, Infinite M200 Pro, Switzerland).

Levels of inflammatory factors and neurotransmitters were measured by Enzyme-linked Immunosorbent Assay (ELISA) in serum and brain tissue, as well as in the colon. Centrifuge the whole blood sample collected in the serum separation tube at 3,000 × g for 15 min, and then stored the supernatant at −80°C. The tissues were raised with pre-cooled PBS (0.01M, pH = 7.4), and cut into pieces after weighing. Put the trimmed tissue into PBS solution (1:9 weight-volume ratio, and added protein inhibitor to PBS) for homogenization, finally centrifuge the homogenate at 5,000 × g for 10 min, and the supernatant was stored at −80°C. A bicinchoninic acid (BCA) Protein Assay kit (SparkJade, China) was used to determine the protein concentration. Levels of IL-6, IL-1β, IL-18 and TNF-α were determined using ELISA Assay kits (JiangLaibio, China) according to manufacturer's instructions. The concentration of 5-HT, DA, NE, HIAA, HVA and DOPAC were determined using the ELISA Assay kits purchased from Jiangsu Meimian Industrial Co, Ltd. (Jiangsu, China) as manufacturer instructions.

Cell supernatants were collected from RAW264.7 and THP-1 macrophages and centrifuged at 3000 rpm at 4°C for 20 min. The concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, inducible nitric oxide synthase (iNOS), and IL-12 were determined with ELISA Assay Kits (MeiMian, Wuhan, China) according to the manufacturer’s instructions. All samples were measured at 450 nm wavelength by an Epoch-2 microplate reader (BioTek, Winooski, VT, USA).

NLRP-3/Cas-1/GSDMD/IL-1β was one of the most important inflammasome-mediated pyroptosis pathways identified recently [14 (link)]. Liver Cas-1, GSDMD, and IL-1β levels were measured via ELISA assay kits obtained from Jiangsu Meimian industrial Co., Ltd. (Yancheng, China). All experimental procedures were quantified under the supplier’s instructions.