Mouse myoblast C2C12 cells were seeded in 6-well plates (5x105 cells per well) in DMEM plus 10% FBS overnight. Mammary tumor cells generated from MMTV-Her2/Neu 16 (link) and MMTV-PyMT mice 15 (link) were cultured overnight in the same media and changed to serum-free DMEM medium for 24 hours. C2C12 cells were treated with CM for 24 hours. For neutralizing antibody assay, CM was pre-incubated with one μg/ml of anti-TNFα antibody (R&D systems, Minneapolis, MN) at room temperature for one hour before adding to C2C12 cells. To directly measure the effects of cytokines on miR-486 expression, C2C12 cells were treated with 20 ng/ml of CCL2, IFNγ, IL-1α, or TNFα (R&D systems) overnight. For promoting differentiation to myotubes, 5000 C2C12 cells were plated in 6-well plates and maintained in 2% of horse serum containing media for seven days. Serum-free control or MMTV-PyMT or MMTV-Her2/Neu tumor cell line-derived CM were added two days after plating.
Anti tnfα antibody
Manufactured by R&D Systems
Sourced in France
The Anti-TNFα antibody is a laboratory-grade reagent that specifically binds and detects Tumor Necrosis Factor alpha (TNFα), a key pro-inflammatory cytokine. This antibody can be used for various research applications involving the measurement and analysis of TNFα.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Il 1α, by R&D Systems (1 mentions)
Z vad fmk, by Enzo Life Sciences (1 mentions)
Ovcar 3 cells, by American Type Culture Collection (1 mentions)
Human tnf α, by R&D Systems (1 mentions)
Actinomycin d, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Quanti blue, by InvivoGen (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Thiazolyl blue tetrazolium bromide mtt, by Merck Group (1 mentions)
Balb c mice, by Charles River Laboratories (1 mentions)
Biotin conjugated goat anti rabbit, by Vector Laboratories (1 mentions)
4 protocols using anti tnfα antibody
1
Tumor-Derived Factors Modulate Myoblast Differentiation
2
Ovarian Cancer Cell Lines Characterization
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SKOV3 cells were obtained from Dr. Robert Mach (Washington University School of Medicine, St. Louis, MO), Hey A8 and Hey A8 MDR cells from Dr. Anil Sood [40 (link)] (M.D. Anderson Cancer Center, Houston, TX), and OVCAR3 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). SKOV3 cells were labeled with a eYFP/luciferase reporter fusion protein by retroviral infection to generate SKOV3-Luc cells (G. Garg and D. Spitzer, unpublished data). Protein expression was confirmed by flow cytometry and in vitro luciferin conversion. SW43 [19 (link)] and SW IV-52 [41 (link)] were synthesized as previously reported. Synthesis of SW IV-134 is described in detail in the Additional file 7 : Supplementary methods. MG-132 was purchased from Calbiochem (Billerica, MA), Z-VAD-FMK from Enzo Life Sciences (Ann Arbor, MI), and anti-TNFα antibody was purchased from R&D systems (Minneapolis, MN).
3
Inflammatory Signaling Pathway Analysis
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Compounds were obtained from Chembridge (San Diego, CA, USA). Dimethyl Sulfoxide (DMSO), Lipopolysaccharide (LPS), TMB, Thiazolyl Blue Tetrazolium Bromide (MTT) and DAB were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). Human TNFα, human TNFβ and anti-TNFα antibody were obtained from R&D Systems (Lille, France). Anti-cleaved caspase 3 was obtained from Cell Signaling Technology (St Quentin en Yvelines, France), Anti-rabbit antibody coupled with HRP was obtained from Abcam (Paris, France) Dulbecco’s Modified Eagle and Phosphate Buffered Saline (PBS) were obtained from Pan Biotech (Brumath, France). Actinomycin D and D-Galactosamine were obtained from Fisher (Illkirch, France). L929 cell line has been grown in the Laboratory for years. HEK-Blue™ TNFα reporter cell line and QUANTI-Blue™ were obtained from InvivoGen (Toulouse, France). 7 weeks-old female Balb/C mice were obtained from Charles River Laboratories (L’Arbresle, France). Mice used in all experiments were handled according to the guidelines and protocols were approved by the ethical commitee of Paris Descartes University, France.
4
Immunohistochemical Analysis of TNF-α Expression
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Brain tissues were fixed in formalin and dehydrated by concentration gradient of ethanol and embedded into paraffin, and then were transversely cut into 5 μm sections, which were subjected to be stained with hematoxylin and eosin. The brain sections were dewaxed by Xylene, then hydrated, after heat antigen retrieval, the sections were labeled with anti-TNF-α antibody (R&D), biotin-conjugated goat anti-rabbit (KPL) as the secondary antibody labeled the primary antibody and combined with Elite AB (Vector), at last DAB were used for color development.
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