Mouse tnf α

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Mouse TNF-α is a cytokine that plays a central role in inflammation and immune responses. It is a key mediator of the inflammatory response and is involved in a wide range of biological processes, including cell proliferation, differentiation, and apoptosis.

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TNF-α (304 citations) Mouse TNF-α ELISA kit (15 citations) Anti-mouse TNF-α (5 citations) Brilliant Violet 421-anti-mouse TNF-α antibodies (2 citations) Mouse TNF-α ELISA Set II (2 citations) Anti-mouse TNF-α (MP6-XT22, (2 citations) ELISA set for mouse IL-6 and TNF-α (2 citations) PE‐labelled rat anti‐mouse TNF‐α antibody (2 citations) Mouse TNF-α and IL-6 ELISA kits (2 citations) Mouse TNF-α (OptEIA-560478). (2 citations)

19 protocols using mouse tnf α

In Vitro Assays for Inflammatory Mediators

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Sepharose CL-6B and Sephadex G-50 were acquired from Pharmacia (Uppsala, Sweden). Trypsin, proteinase K, α-chymoTrypsin, and lipase were purchased from Sigma (St. Louis, MO, USA). Lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), Griess reagent, and 3-(4,5-dimethylthiazol-2-2,5-diphenyltetrazolium bromide (MTT) were also purchased from Sigma. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and a penicillin-streptomycin solution were acquired from Invitrogen (Grand Island, NY, USA). A rabbit anti-mouse iNOS polyclonal antibody was purchased from Santa Cruz Biotech Inc. (Santa Cruz, CA, USA), and an anti-mouse COX-2 antibody from Cayman Co. Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG antibody and anti-mouse IgG antibody were purchased from Cell Signaling Technology (Danvers, MA). Alkaline-phosphatase-conjugated donkey anti-mouse IgG antibody was purchased from Jackson Immunoresearch Laboratories Inc. Mouse TNF-α, IL-6, IL-1β, and an enzyme-linked immunosorbent assay (ELISA) kits were purchased from BD Biosciences (San Diego, CA, USA). All other chemicals and reagents were of analytical grade.

Choi Y.H., Cho S.S., Simkhada J.R., Rahman M.S., Choi Y.S., Kim C.S, & Yoo J.C. (2017). A novel multifunctional peptide oligomer of bacitracin with possible bioindustrial and therapeutic applications from a Korean food-source Bacillus strain. PLoS ONE, 12(5), e0176971.

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Cytokine Quantification in Activated Cells

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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).

Park Y., Dodantenna N., Kim Y., Kim T., Lee H., Yoo Y., Heo J., Lee J., Kwon M., Kang H.C., Lee J, & Cho H. (2023). MARCH5‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation. The EMBO Journal, 42(19), e113481.

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Cytokine and Adhesion Molecule Assay

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Recombinant mouse IL-23, E-selectin and P-selectin Fc-chimeras were from R&D Systems (Minneapolis, MN). Recombinant mouse IL-12, IL-2, IL-6, TNF-α, recombinant human TGF-β, and the following antibodies to mouse cytokines and adhesion molecules: IL-4 (clone 11B11), IFNγ (clone XMG 1.2), IL-2 (clone JES6-1A12), CD4 (clone GK 1.5), CD3 (clone145-2C11), CD28 (clone 37.51), IL-17A (clone 2C11-18H10.1), CD43 activation-associated glycoform (clone 1B11), and CD44 (clone IM7) are all from Biolegend (San Diego, CA). Anti- PSGL-1 and mouse TNF-α were purchased from BD-Pharmingen (San Jose, CA), and carrier free CCL20 from Peprotech (Rocky Hill, NJ). PMA and ionomycin were from SIGMA (St. Louis, MO). Secondary Abs coupled to alkaline phosphatase were from Promega (Madison, WI). Vibrant CFSE and Alexa 680 were from Life Technologies (Carlsbad, CA). Myelin Oligodendrocyte glycoprotein was purchased from Anaspec (Fremont, CA) and Pertussis Toxin was purchased from List Biological Laboratories (Campbell, CA). Anti-E-selectin (clone 9A9) antibody was generously provided by Dr. F. William Luscinskas (Brigham and Women's Hospital, Boston, MA) and IgG control was from Biolegend (San Diego, CA).

Velázquez F., Grodecki-Pena A., Knapp A., Salvador A.M., Nevers T., Croce K, & Alcaide P. (2015). CD43 functions as an E-selectin ligand for Th17 cells in vitro and is required for rolling on the vascular endothelium and Th17 cell recruitment during inflammation in vivo. Journal of immunology (Baltimore, Md. : 1950), 196(3), 1305-1316.

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Cytokine Profiling Assay for Mouse Samples

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A proteome profiler array (mouse cytokine array panel A; R&D Systems, Minneapolis, MN) was used to assay the level of 40 cytokines according to the manufacturer’s instructions. Briefly, the capture antibody of cytokines and chemokines were spotted on nitrocellulose membranes. The sample/antibody mixture was incubated, and the cytokine-chemokine antibody complex was bound to the immobilized capture antibody on the membrane. After the streptavidin-horseradish peroxidase and chemiluminescent reagents had been added, the signal was proportional to the amount of cytokine bound. For secreted cytokines, supernatants were collected from infected/treated cells at the indicated times. Mouse TNF-α (BD Biosciences/Pharmingen) and mouse MCP-1 (R&D Biosystems) were measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions.

Das S., Sarkar A., Choudhury S.S., Owen K.A., Derr-Castillo V.L., Fox S., Eckmann L., Elliott M.R., Casanova J.E, & Ernst P.B. (2015). Engulfment and Cell Motility Protein 1 (ELMO1) Has an Essential Role in the Internalization of Salmonella Typhimurium Into Enteric Macrophages That Impact Disease Outcome. Cellular and Molecular Gastroenterology and Hepatology, 1(3), 311-324.

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Splenocyte Stimulation Assay

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Spleens from C57BL/6 mice were immediately excised upon killing. Single-cell suspensions were achieved in vitro via mechanical dissociation. Red blood cells were lysed by resuspension in ammonium-chloride-potassium lysis buffer and incubating at room temperature for 2 min. Cells were resuspended at a concentration of 1 × 10⁶ cells/mL in Iscove’s modified Dulbecco’s medium (Gibco) supplemented with 5% FBS, 1% penicillin-streptomycin, and 40 μmol/L 2-mercaptoethanol. Splenocytes were restimulated with CSDE1, CSDE1^P5S, TYRP2, or VSV-N_52-59 peptides with or without the addition of CD200AR-L or control SIINFEKL (5 μg/mL). Supernatants were collected and assayed for IFN-γ by enzyme-linked immunosorbent assay (ELISA) as per the manufacturer’s instructions (Mouse TNF-α or Mouse IFN-γ ELISA Kit, OptEIA, BD Biosciences).

Webb M.J., Kottke T., Kendall B.L., Swanson J., Uzendu C., Tonne J., Thompson J., Metko M., Moore M., Borad M., Roberts L., Diaz R.M., Olin M., Borgatti A, & Vile R. (2023). Trap and ambush therapy using sequential primary and tumor escape-selective oncolytic viruses. Molecular Therapy Oncolytics, 29, 129-142.

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Cytokine Secretion Profiling by ELISA

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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).

Chathuranga K., Kim T., Lee H., Park J., Kim J., Chathuranga W.A., Ekanayaka P., Choi Y.J., Lee C., Kim C., Jung J.U, & Lee J. (2020). Negative regulation of NEMO signaling by the ubiquitin E3 ligase MARCH2. The EMBO Journal, 39(21), e105139.

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Evaluation of Hepatic Oxidative Stress

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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).

Lee J.A., Shin M.R., Choi J., Kim M., Park H.J, & Roh S.S. (2022). Co-Treatments of Gardeniae Fructus and Silymarin Ameliorates Excessive Oxidative Stress-Driven Liver Fibrosis by Regulation of Hepatic Sirtuin1 Activities Using Thioacetamide-Induced Mice Model. Antioxidants, 12(1), 97.

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Cytokine Profiling in Colonic Tissue

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Cytokines level in colonic tissue homogenates and culture supernatant were measured by mouse TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-12p40, and IL-17A ELISA kits (BD Pharmingen, San Diego, CA, USA), mouse IL-1β, IL-5, IL-22, IL-23 ELISA kits (Thermo Fisher Scientific), mouse OSM, IL-13 ELISA kits (R&D, Minneapolis, MN, USA), and human CXCL10 kits (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. Cytokines in serum were quantified by Luminex x-MAP technology (Luminex Corp, Austen TX, USA) on a Luminex 200 instrument (Merck Millipore, Billerica, MA, USA). All standard curves with four-parameter logistic fitting, supplied by the manufacturer, had R² values at or close to 1. Cytokines concentration were obtained from the standard curves.

Li H., Feng C., Fan C., Yang Y., Yang X., Lu H., Lu Q., Zhu F., Xiang C., Zhang Z., He P., Zuo J, & Tang W. (2020). Intervention of oncostatin M-driven mucosal inflammation by berberine exerts therapeutic property in chronic ulcerative colitis. Cell Death & Disease, 11(4), 271.

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Antigen-Specific T Cell Activation by PS-Loaded PLGA Nanoparticles

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Mouse T cells were purified from spleens of 2D2 mice, a genetically engineered mouse model with only T cells expressing T cell receptors to MOG, using anti-CD4⁺ beads (MACS) [45 (link)]. Bone-marrow derived DCs were incubated with PS-loaded PLGA nano-particles (12, 25, or 50 μM PS), PS alone (12, 25, or 50 μM), or blank PLGA nanoparticles at 37 °C for 4 h, and the cells were subsequently washed. For myelin-specific T cell activation, 5 × 10⁴ DCs treated with PS-loaded PLGA nanoparticles, PS alone, or blank PLGA nanoparticles at 37 °C for 4 h were pulsed with MOG_35–55 peptide (50 μg/ml) and then incubated with 2 × 10⁵ T cells for 48 h. The cells and media were separated, and the supernatant was probed for mouse IFNγ (Biolegend, 430806), mouse IL-2 (BD, 555148), mouse IL-6 (BD, 555240) mouse TNFα (BD, 555268). T-cells were stained with IL-10 (eBioscience, 12-7101) and FOXP3 (eBioscience, 17-5773-82) to identify the Treg population using flow cytometry.

Roberts R.A., Eitas T.K., Byrne J.D., Johnson B.M., Short P.J., McKinnon K.P., Reisdorf S., Luft J.C., DeSimone J.M, & Ting J.P. (2015). Towards programming immune tolerance through geometric manipulation of phosphatidylserine. Biomaterials, 72, 1-10.

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Cytokine Detection in Cell Culture

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Enzyme-linked immunosorbent assay (ELISA) was performed to detect secreted inflammatory cytokines in cell culture supernatants. Human IL-6 (BD Biosciences, 555220), human interferon-β (CUSABIO, CSB-E09889h), mouse IL-6 (BD Biosciences, 555240), mouse interferon-β (CUSABIO, CSB-E04945m), mouse interferon-α (PBL Assay Science, 42120-1), mouse TNF-α (BD Biosciences, 555268), porcine IL-6 (R&D Systems, P6000B), and porcine IFN-α (CUSABIO, CSB-E07328p) were used for analysis according to the manufacturer’s protocols.

Ekanayaka P., Shin S.H., Weeratunga P., Lee H., Kim T.H., Chathuranga K., Subasinghe A., Park J.H, & Lee J.S. (2021). Foot-and-Mouth Disease Virus 3C Protease Antagonizes Interferon Signaling and C142T Substitution Attenuates the FMD Virus. Frontiers in Microbiology, 12, 737031.

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