Ifn β elisa kit

Antibodies specifically against Runx3 (Cat. No. 9647 and 13089), Runx2, TLR3, MDA5, IKKα, IKKβ, PKD3, cleaved caspase-3, −8, −9, cleaved PARP (Asp214), Stat1, phospho-IKKα (Ser176)/IKKβ (Ser177), phospho-p65 (Ser-536) were from Cell Signaling Technology. Runx1 and NFκB p65 antibodies were from Santa Cruz Biotechnology. RIG-1 antibody was from OriGene Technology. Actin antibody, actinomycin D and LPS (Escherichia coli 0111:B4) were from Sigma. TLR ligands Type B CpG oligonucleotide (ODN 2006), low and high molecular weight poly(I:C), R837, CL075, and Pam3CSK4 were from InvivoGen. Recombinant TGFβ, TNFα, and specific peptide inhibitors for caspase-3 (Z-VAD-FMK), caspase-8 (Z-IETD-FMK) and caspase-9 (Z-LEHD-FMK) were from R & D Systems. IFNβ ELISA kit and IFNα were from PBL Assay Science, and IFNγ and IFNλ were from eBioscience. TUNEL Apo-Green detection kit was from Biotool. Bay 11-7082, Piceatannol and GFX109203X were from EMD Millipore. Amlexanox, SC514 and necrostatin-1 were from Tocris Bioscience. Clarity Western ECL substrate was from Bio-Rad.

Lysozyme (Sigma #L6394), LPS (Sigma #L2630), Glutamate assay kit (Sigma #MAK004) and Minocycline were purchased from Sigma. TLR4 (Abcam #ab22048), PGP9.5 (Abcam #ab108986), GOT (Abcam #ab221939) and Glutaminase (Abcam #ab156876) antibodies were purchased from Abcam. NFκB p65 (CST #8242), Glutamate dehydrogenase (CST #80063), GOT1 (CST #34423), and GOT2 (CST #71692) antibodies were purchased from Cell Signaling Technology. PEPinh MyD, PEPinh TRIF were purchased from Invivogen. Cu-CPT22 (#4884), TAK-242 (#6587), and Dynasore (#2897) were purchased from Tocris. TRIF siRNA (Thermo, #4457308), GOT2 siRNA (Thermo, #4457308), HMGB1 (Thermo #34-8401-82), IFNα mouse ELISA kit (Thermo #BMS6027) and IL-10 mouse ELISA kit (Thermo #BMS614) were purchased from ThermoFisher Scientific. IFN-β ELISA kit (PBL Assay Science #424001) was purchased from PBL Assay Science and mouse inflammatory cytokines ELISA array kit (Qiagen #336161) was purchased from Qiagen. Cell culture media and reagents were purchased from Thermo (Invitrogen). All other reagents were purchased from Sigma until stated otherwise.

An enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of IFN-α and IFN-β in the spinal cord. Mouse high-sensitivity IFN-α ELISA kit and IFN-β ELISA kit were purchased from PBL Assay Science. Spinal tissues were homogenized in a lysis buffer containing protease and phosphatase inhibitors. Tissue samples were centrifuged at 12,500 ×g for 10 min and the supernatant was collected. BCA Protein Assay (Pierce) was employed to determine protein concentrations. For each reaction in a 96-well plate, 100 μg of proteins of samples were used. All ELISA experiments followed the manufacturer’s protocol. The optical densities of samples were measured using an ELISA plate reader (Bio-Rad) and the levels of IFN-α and IFN-β were calculated using the standard curves and ELISA results were reported normalized to protein concentration (pg/mg spinal tissue).

Mouse high-sensitivity IFN-α ELISA kit (42115-1) and IFN-β ELISA kit (42410-1) were purchased from PBL Assay Science. Mouse CTX-I ELISA kit (AC-06F1) and mouse PINP ELISA kit (AC-33F1) were purchased from Immunodiagnostic Systems. ELISA was performed using culture medium, serum, and bone marrow lysates. Serum was obtained from whole blood that was collected from a submandibular vein via facial vein puncture, coagulated for 30 min at room temperature, followed by centrifugation (2000 × g for 10 min, 4 °C) and collection of the supernatant (serum). Bone marrow was homogenized in a lysis buffer containing protease inhibitors at 4 °C for 30 min. ELISA was conducted in accordance with the manufacturer’s instructions. A standard curve was performed for each experiment.

For analysis of cytokine production, supernatants were harvested after overnight stimulation and stored at –20ᵒC. IFN‐β and CXCL10 cytokine production after stimulation with Poly I:C was determined by harvesting the supernatants 3 h after stimulation and supernatants were again stored at –20ᵒC. Cytokine levels in supernatants were measured by ELISA, using an IFN‐β ELISA kit (PBL Assay Science), antibody pairs for CXCL10 (R&D Systems), TNF (MAb1; MAb11; eBioscience), and IL‐1β (CT213‐c; CD2013‐d; U‐CyTech).