Protein homogenates were prepared from freshly isolated spinal cords and whole brains and mechanically homogenized in 1 mL of 1× PBS containing protease inhibitors (Pierce Protease Inhibitor Tablets, Thermo Fisher Scientific, Grand Island, NY). Total protein content was measured using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific), following the manufacturer’s instructions. Homogenates were subsequently centrifuged at 20,000×g for 20 min at 4 °C. Supernatants were collected and diluted, and 100 μL was loaded into sandwich ELISA plates following the manufacturer’s protocol. Mouse IL-1β, IL-6, IL-17, IFN-γ, IFN-β, and TNF-α DuoSet ELISA kits (R&D Systems) were used. The RayBio Mouse Axl ELISA kit was used to detected protein levels of Axl following the manufacturer’s protocol (RayBiotech, Norcross, GA).
Tnf α duoset elisa kits
Manufactured by R&D Systems
The TNF-α DuoSet ELISA kits provide the components necessary for the development of sandwich enzyme-linked immunosorbent assays (ELISA) for the quantitative measurement of human Tumor Necrosis Factor alpha (TNF-α) in cell culture supernatants, serum, and plasma.
Automatically generated - may contain errors
Lab products found in correlation
Pierce protease inhibitor tablet, by Thermo Fisher Scientific (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Amplification grade dnase 1 kit, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Accutase, by Merck Group (1 mentions)
2 propanol, by Merck Group (1 mentions)
Human il 1β, by R&D Systems (1 mentions)
Toxilight assay kit, by Lonza (1 mentions)
Nuclease free water, by Qiagen (1 mentions)
Primers for qpcr, by Eurofins (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
7 protocols using tnf α duoset elisa kits
1
Quantification of Cytokines and Axl Protein
2
Cell Culture and Cytokine Analysis Protocol
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RPMI-1640 media, 2-mercaptoethanol (ME), Hank’s Balanced Salt Solution containing MgCl2 and CaCl2 (HBSS), sodium pyruvate, and TRIzol® reagent were purchased from Thermo Fisher Scientific. Fetal calf serum (FCS), bovine serum albumin (BSA), Penicillin/Streptomycin (P/S), D-glucose, trypsin, accutase, phorbol 12-myristate-13-acetate (PMA), LPS, endotoxin-free H2O, phosphate-buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-propanol, and the amplification grade DNase I Kit were purchased from Sigma-Aldrich/Merck. The ToxiLight assay kit was purchased from Lonza. Ethanol and sulfuric acid were purchased from Roth. Nuclease-free water was purchased from Qiagen. The iScript™ cDNA Synthesis Kit and the iQ™ SYBR® Green Supermix were purchased from Bio-Rad. Primers for qPCR were purchased from Eurofins. The Human IL-1β, IL-6, IL-8, and TNFα DuoSet ELISA Kits were purchased from R&D Systems.
3
Cytokine Profiling in Corpus Callosum
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Protein homogenates were prepared from freshly isolated corpora callosa and mechanically homogenized in 1 mL of 1 × PBS containing protease inhibitors (Pierce Protease Inhibitor Tablets, ThermoFisher Scientific). Total protein content was measured using a NanoDrop. Homogenates were subsequently spun down at 20,000 × g for 20 min at 4°C. Supernatants were collected and 100 μL were loaded into sandwich ELISA plates following manufacturer's protocol. Mouse IL-6, IL-12 p70, IFN-γ, IFN-β, IL-17, TNF-α DuoSet ELISA kits (R&D Systems, Minneapolis, MN) were used.
4
Lipid-based Nanoparticle Formulations for Immunological Studies
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DOTAP, 1,2-distearoryl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol-2000) ammonium salt (DSPE-PEG2000) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol and protamine sulfate salt from salmon (Grade X) were purchased from Sigma-Aldrich (St. Louis, MO). Collagenase/dispase and DNase I were purchased from Roche diagnostics. Fluorescent antibodies against mouse B220 (clone RA3-6B2), CCR7 (clone 4B12), CD4 (clone GK1.5), CD8a (clone 53–6.7), CD11b (clone M1/70), CD11c (clone N418), CD44 (clone IM7), CD62L (clone MEL-14), CD69 (clone H1.2F3), CD80 (clone 16-10A1), CD86 (clone GL-1), CD103 (clone 2E7), CD127 (clone A7R34), CD138 (clone 281–2), GL-7 (clone GL7), IFN-γ (clone XMG1.2), IL-2 (clone JES6-5H4), Ki67 (clone SolA15), KLRG1 (clone 2F1/KLRG1), Ly6G (clone 1A8), MHC-II (clone M5/114.15.2), NK1.1 (clone PK136), PD-1 (clone 29F.1A12), PDCA1 (clone 927), TIM-3 (clone 8B.2C12), TNF-α (clone MP6-XT22), purified CD16/32 (clone 93), and brefeldin A were from BioLegend (San Diego, CA). PNA and Lipofectamine 2000 were from Thermo Fisher Scientific. The mouse IL-1β, IL-2, IL-6, IL-10, IFN-γ, and TNF-α DuoSet ELISA kits were purchased from R&D Systems (Minneapolis, MN).
5
Murine Immunology Antibody Panel
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Anti–γ-interferon (IFN-γ), –interleukin (IL)-2, –IL-10, and –IL-17A antibody pairs for ELISA; fluorochrome-conjugated anti-CD4, -CD8, -B220, -Vβ4, -Vβ5, -Vβ8, -CD11b, and -CD11c antibodies for fluorescence-activated cell sorter (FACS); and anti-CD3ε and anti-CD28 antibodies were purchased from BD Biosciences. IL-1β, CCL5, and tumor necrosis factor (TNF)-α DuoSet ELISA kits, CXCL10 antibody pairs, and fluorochrome-conjugated anti–IL-12Rβ2 were purchased from R&D Systems. Fluorochrome-conjugated anti-CD19, -CD25, -CD44, and -CD69 antibodies were purchased from eBioscience, while biotin anti-mouse CD4, in addition to anti-F4/80 fluorochrome-conjugated and live/dead fluorochrome-conjugated antibodies, was purchased from Invitrogen. Anti–P-STAT4 (Y693) antibody was purchased from Cell Signaling, while anti-STAT4 antibody was obtained from Biolegend. The BDC-2.5 mimotope (EKAHRPIWARMDAKK) was synthesized by the University of Alabama at Birmingham peptide synthesis core facility. Xanthine oxidase and β-actin were obtained from Sigma-Aldrich, and r-hIL-2 was purchased from Peprotech.
6
ELISA Quantification of IL4 and TNFα
7
Cytokine Secretion Measurement via ELISA
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For measurement of secreted cytokines using Mouse IL-10, IL-6 and TNF-α DuoSet ELISA kits (R&D Systems Europe – Biotechne), culture supernatants were collected and stored at −20°C.
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