Mouse il 6

To measure type-I interferon and proinflammatory cytokines, mouse IFN-β (PBL interferon source), mouse IL-6 (BD Biosciences), human IFN-β (PBL interferon source) and human IL-6 (BD Biosciences) ELISA kits were used. After infection of virus to cells, the levels of secreted cytokines were measured in supernatant of infected cell. For mice, plasma levels of type-I interferon and proinflammatory cytokines were analysed.

Cells were seeded into 12‐well plates and treated with indicated activators and indicated bacteria. The supernatant was collected at the indicated time points and then quantified by using following commercially available ELISA kits: mouse IL‐1β (Biolegend, 432616), Caspase‐1 (Novus biologicals, NBP2‐75014), mouse TNF‐α (BD OptEIA, 555268), mouse IL‐6 (BD OptEIA, 555240), and mouse IL‐18 (MBL, 065FA).

At the end of the animal experiments, serum were collected and levels of specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA).
Serum titers of anti-CII or anti-SRBC IgG were determined as previously described51 (link)54 (link). Briefly, 96-well ELISA plate were coated with bovine CII (50 μg/ml) or extracted SRBC membranes (20 μg/ml) (prepared as described previously55 (link)) overnight at 4 °C. Each diluted serum sample was added and incubated. Horse-radish peroxidase (HRP) conjugated goat anti-mouse IgG (H + L), IgG1and IgG2a Abs (Invitrogen, San Diego, CA, USA) were used. The optical density is measured spectrophotometrically at 450 nm.
Cytokines in sera and culture supernatants were detected using mouse IL-6, IL-17A (BD Biosciences, San Diego, CA) and IL-21 (R&D systems, Minneapolis, MN) ELISA kits according to the manufacturer’s instructions.

ELISA was performed to detect the secreted IFNs and proinflammatory cytokines in cell culture supernatants. Mouse IL-6 (BD Biosciences, 555240), mouse interferon-β (CUSABIO, CSB-E04945m), human IL-6 (BD Biosciences, 555220), human interferon-β (CUSABIO, CSB-E09889h), porcine IL-6 (R&D Systems, P6000B), and porcine interferon-β (CUSABIO, CSB-E09890p) were used for the analysis according to the manufacturer’s protocols.

Mouse sera, tissue homogenates, or cell supernatants were used to examine level of cytokine secretion by ELISA. Commercial kits as followings were used according to manufacturer's protocols: mouse IFN‐β (PBL Interferon Source), mouse IL‐6 (BD Biosciences), mouse IL‐12 (BD Biosciences), mouse TNF‐α (BD Biosciences), mouse CCL5 (R&D Systems), mouse CXCL10 (R&D Systems), mouse IL‐1β (BD Biosciences), human IFN‐β (PBL interferon source), and human IL‐6 (BD Biosciences).

TBARS assay to evaluate malondialdehyde (MDA) was estimated using commercially available kit (BioAssay System, Hayward, CA, USA). Hepatic ROS levels were measured using DCF_DA fluorescence probe. Briefly, small pieces of hepatic tissue were homogenized in 10 mM PBS (pH.7.2) and centrifuged at 3000× g, 20 min, 4 °C. Then supernatants were collected and transferred 100 µL of sample lysates to black wall of 96-well microplate and 10 µL of DCF_DA 10 µM were added to the plate. Plate was incubated in 37 °C for 20 min and fluorescents were measured under the excitation/emission at 485 nm/535 nm of wavelengths. Results appeared as fold changes, which were normalized by control group. Hepatic protein levels of pro-inflammatory cytokines were measured using commercially available kits according to the manufacturer’s protocol (mouse TNF-α and mouse IL-6 for BD Bioscience, San Jose, CA, USA; mouse IL-1β for R&D system, Minneapolis, MN, USA).