Elisa kits for tnf

Mouse monoclonal antibodies against Flag (Origene, 1:2000, F3165), HA (Origene, 1:2000, H6908), β-actin (Sigma, 1:10,000, A2228), Myc (CST, 1:1000, 5605), p-IκBα (CST, 1:1000, 9246 L); rabbit antibodies against p-IRF3 (CST, 1:500, 37829), USP19 (Abcam, 1:1000, ab93159), TRIF (Abcam, 1:1000, ab180689), p65 (Santa Cruz Biotechnology, 1:1000, 71675), p-p65(S536) (CST, 1:1000, 3033),TBK1 (Abcam, 1:1000, ab40676) and p-TBK1 (Abcam, 1:1000, ab109272), ubiquitin (Abcam, 1:500, ab134953), K27-linkage specific polyubiquitin (Abcam, 1:1000, 181537), TLR3 (CST, 1:500, 6961), TLR4 (R&D, 1:500, AF1478), TRAM (Abcam, 1:500, ab96106), KCTD10 (Proteintech, 1:1000, 27279–1-AP); poly(I:C) (Invivogen), LPS (Sigma), R848 (Invivogen), PGN (Invivogen), human IFN-γ (Peprotech), murine M-CSF (Peprotech), Trizol (Takara Bio), SYBR Green (BIO-RAD), dual-specific luciferase assay kit (Promega, E1980), polybrene (Millipore,TR-1003-G), type II collagenase (Worthington), DNase I (Sigma-Aldrich), and d-galactosamine hydrochloride (D-Gal) (Sigma); and ELISA kits for TNF (Biolegend), IL-6 (Biolegend), CXCL10 (Boster) and IFN-β (PBL) were purchased from the indicated companies. HEK293 cells were obtained from ATCC. SeV (Cantell strain) (Charles River Laboratories), HSV-1 (KOS strain) (China Center for Type Culture Collection, Wuhan, China) were obtained from the indicated companies.

Cytokine determination in the preserved supernatants was performed using the ELISA kit, TNF-α and IL-6, according to the attached protocol. Cytokine levels in the supernatants were determined using Biolegend ELISA kits for TNF-α and IL-6 according to the manufacturer’s protocols. The optical density was measured using a Multiskan RC spectrophotometric reader (Thermo Labsystems, Waltham, MA, USA) at λ = 450 nm.

Cytokines levels were determined using ELISA kits for TNF, IL6, GM-CSF (all Biolegend), CXCL2, CXCL1, IL-12B, LCN2 (all Thermo Fisher Scientific) according to the manufacturer’s instructions.