Monocyte-derived IL-15 DCs were prepared conforming to our previously described 48-hour culture protocol (18 (link), 20 (link)). Positively selected CD14+ monocytes (Miltenyi) are cultured in Roswell Park Memorial Institute medium (Life Technologies, Merelbeke, Belgium) with 2.5% heat-inactivated human AB serum (Life Technologies) at a final concentration of 1–1.2 × 106 cells/mL. To generate mature IL-15 DCs, a 28-hour differentiation step using GM-CSF (800 IU/mL; Life Technologies) and IL-15 (200 ng/mL; Immunotools, Friesoythe, Germany) is followed by maturation induction with R848 (3 µg/mL; Alexis Biochemicals, San Diego, USA), interferon (IFN)-γ (250 ng/mL; Immunotools), tumor necrosis factor (TNF)-α (2.5 ng/mL; Life Technologies), and prostaglandin E2 (1 µg/mL; Pfizer, Puurs, Belgium) for 20 hours. To collect 48-hour wash-out supernatant, IL-15 DCs are harvested, thoroughly washed, and reseeded in fresh medium at a concentration of 1 × 106 cells/mL in low-absorbing polypropylene tubes. After 48 hours, cell-free supernatant is collected and frozen at −20°C, until further use.
Tumor necrosis factor α
Manufactured by Thermo Fisher Scientific
Sourced in United States
Tumor necrosis factor (TNF)-α is a cytokine that plays a crucial role in the regulation of immune cells and the inflammatory response. It is involved in the signaling pathways that mediate cell death, proliferation, and differentiation. TNF-α is commonly used in research applications to study its biological functions and interactions within cellular systems.
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Lab products found in correlation
Interferon ifn γ, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Interleukin il 1β, by R&D Systems (3 mentions)
Interleukin 6 (il 6), by R&D Systems (3 mentions)
Plasminogen, by Enzyme Research (3 mentions)
Gm csf, by Thermo Fisher Scientific (2 mentions)
Recombinant human interleukin il 1β, by Thermo Fisher Scientific (2 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions)
Il 1β, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Il 10, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Neutrophil elastase, by R&D Systems (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Cd14 monocytes, by Miltenyi Biotec (1 mentions)
Roswell park memorial institute medium, by Thermo Fisher Scientific (1 mentions)
Il 15, by Immunotools (1 mentions)
Interferon ifn γ, by Immunotools (1 mentions)
Prostaglandin e2, by Pfizer (1 mentions)
31 protocols using tumor necrosis factor α
1
Generation of Monocyte-derived IL-15 DCs
2
Cytokine and Metabolite Assays
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Tumor necrosis factor (TNF)-α was purchased from Life Technologies. Interleukin (IL)-1β, IL6, transforming growth factor (TGF)-β, IGF1 and IGF2 were purchased from R&D Systems. Reagents for SDS-PAGE, mini-gels and blocking buffer were purchased from Bio-Rad Laboratories. Mannitol and d-glucose were purchased from Sigma-Aldrich. Advanced glycation end products (AGE), AGE
3
3D Co-culture of PBMC and MSC
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3D co-cultures of PBMC with MSC (n = 6) were performed in 12-well plates (Corning). Cells were embedded in fibrin matrices in a ratio 1:100 (MSC:PBMC) using 2.5 × 106 PBMC/cm2 and cultured for up to 2 weeks in complete αMEM medium (Invitrogen) containing 10% fetal bovine serum (GE Healthcare Life Sciences). Fibrin matrices were prepared as described above, but without addition of aprotinin. Control experiments were performed culturing PBMC separated from MSC by a 0.4 μm transwell insert (Corning), PBMC without support of stromal cells and MSC alone. To investigate the effect of paracrine inflammatory signals on stromal cells in the absence of immune cells, MSC (n = 4) were embedded in fibrin matrices in 24-well plates (Corning) at 2.5 × 104 cells/cm2 and cultured for 6 days in complete αMEM medium (Invitrogen) containing 10% fetal bovine serum (GE Healthcare Life Sciences) supplemented with 5 ng/ml tumor necrosis factor (TNF)α (PeproTech, Rocky Hill, NJ) and 10 ng/ml interferon (IFN)γ (PeproTech). Control experiments were performed in complete αMEM medium without cytokine supplementation. Cultures were maintained at 37°C (20% O2 and 5% CO2 humidified atmosphere), and medium was changed every 3 days. Cellular re-arrangement was monitored using a phase contrast microscope (Olympus) and documented using a digital camera (Olympus).
4
Peptide Production and Characterization
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Chemically synthesized AMP peptide (LDALVKEKKLQGKGPGGPPPK), a scrambled peptide (GKPLGQPGKVPKLDGKEPLAK), and recombinant human (rh)AMP-18 were prepared by GenScript (Piscataway, NJ) as described previously [13 (link)]. Briefly, rhAMP-18 is expressed and purified as a His6-tagged fusion protein. The coding sequence for full-length human AMP-18 was cloned into an E. coli expression vector, pGSE3, and the expressed protein was purified from 5 L of culture medium by affinity column chromatography. AMP peptide and rhAMP-18 were found to be equally effective in triggering cellular responses as previously reported [13 (link)–15 (link)]. Cell culture medium, fetal bovine serum (FBS), penicillin and streptomycin were obtained from Gibco BRL, Life Technologies (Gaithersburg, MD). Tumor necrosis factor (TNF)-α was purchased from PeproTech (Rocky Hill, NJ), and other reagents from Sigma-Aldrich unless otherwise specified.
5
Cibinetide Dosage Optimization for In Vitro and In Vivo Studies
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Cibinetide was provided by Araim Pharmaceuticals, Inc. (Tarrytown, NY, USA). Cibinetide stock solution (1.2 mg/mL, 1 mmol/L) was dissolved in phosphate buffered saline (PBS), aliquoted and stored at -20°C until use. The doses of cibinetide (100 nmol/L in in vitro study and 120 µg/kg in in vivo model) were selected based on our previous studies21 (link),22 (link). Recombinant human interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were purchased from PeproTech Inc., (Rocky Hill, NJ, USA).
6
Isolation and Culture of Mouse Vascular Smooth Muscle Cells
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Mouse VSMCs were isolated from the aortas of male 8-week-old C57BL/6J mice as reported previously 18 (link), 19 (link). Cells were cultured in DF12 (Dulbecco’s modified Eagle’s medium and Ham’s F-12 nutrient mixture) containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2. Cells at <5 passages were used in all cell experiments. For related experiments, cells were treated with 10 ng/ml tumor necrosis factor (TNF)-α (PeproTech, Rocky Hill, New Jersey) for 24 h or TNF-α plus 5 μmol/ml of 5-aza (MilliporeSigma, St. Louis, Missouri) for 48 h. In a CD137-associated assay, VSMCs were pre-treated with TNF-α (10 ng/ml) for 24 h to elevate the CD137 levels on the membrane and further treated with 10 μg/ml recombinant CD137L (Sangon Biotech, Shanghai, China) to activate CD137 signaling (20) (link).
7
Cytokine Profiling of Splenic MNCs
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On day 28 p.i., mice were sacrificed and spleens were removed under aseptic conditions. Splenic MNCs (6 × 105/mL) were cultured for 48 hour at 37°C in the presence of MOG35‐55 (10 μg/mL). Supernatants were collected and measured for cytokine concentrations of IL‐17, IL‐10 (eBioscience Inc), IL‐6, tumor necrosis factor (TNF‐α) (PeproTech Inc., Hawthorne, NJ, USA) and IL‐1β (Invitrogen Inc., Carlsbad, CA, USA) using sandwich enzyme linked immunosorbent assay (ELISA) kits in accordance with the manufacturer's instructions. The quantitation of cytokines was calculated by reference to standard curves. Determinations were performed in triplicate and results were expressed as pg/mL.
8
Murine Lung Epithelial and Macrophage Response to Hypoxia and TNF-α
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Murine lung epithelial (MLE15) and murine alveolar macrophage (MH-S) cells were plated on 12-well plates (2.5 × 105 cells per well ) and cultured in RPMI-1640 (Nacalai Tesque, Tokyo, Japan) supplemented with 4 and 10 % fetal bovine serum (FBS), respectively, for 24 h. The cells were then FBS-starved for 12 h. Subsequently, the culture medium was exchanged to FBS-free medium with or without 500 pg/mL tumor necrosis factor (TNF)-α (PeproTech, Rocky Hill, NJ) to simulate a sterile inflammatory environment, and the cells were cultured in either 21 % (nonhypoxic condition) or 5 % (hypoxic condition) O2 by using CulturePal5 (Mitsubishi Gas Chemical Company, Tokyo, Japan) for 24 h. Conditioned medium was collected and centrifuged at 3000g for 10 min at 4 °C. The supernatants were aliquoted and stored at −80 °C until use. Finally, the cells were lysed for either protein or RNA extraction.
9
Generating Mature Dendritic Cells
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DCs were generated as previously described.27 (link) Briefly, bone marrow cells obtained from mouse femurs were cultured in 6-well plates (5×106 cells/well) in culture medium supplemented with 40 ng/mL granulocyte macrophage-colony stimulating factor and 40 ng/mL IL-4 (both from CreaGene, Seongnam, South Korea). After 3 days, the non-adherent cells were removed by gently shaking the dish and then replacing the medium. On day 4, the non-adherent cells were again removed by the same method. On day 6, half the culture medium was replaced with fresh medium. To induce maturation, DCs were exposed to 50 ng/mL interferon (IFN)-γ and 50 ng/mL tumor necrosis factor (TNF)-α (both from PeproTech Inc., Rocky Hill, NJ, USA), or treated with the indicated NPs for 48 h. The DCs were harvested by gentle pipetting on day 8.
10
Mouse Inflammation Induction and Inhibition
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All experiments were performed using 8- to 12-week-old mice. Depending on the experiment 0.25 mg/kg of LPS (Escherichia coli 0111:B4; Sigma), 0.75 mg/kg of tumor necrosis factor (TNF)-α (PeproTech), 0.25 mg/kg of interleukin (IL)-1α (PeproTech), or 0.25 mg/kg of IL-1β (PeproTech) dissolved in 200 μL of PBS were injected IP, unless stated differently. Treatment lasted for 18 hours unless indicated differently. Control mice were injected with 200 μL of PBS. TNFα inhibition was induced by intraperitoneal injection of etanercept (EnbrelO, FDA 1998) daily for 10 days (5 mg/kg in 100 μL of PBS on days 1-5; 7.5 mg/kg in 150 μL of PBS on days 6-10). To block IL-1 signaling, 2.5 mg/kg of anakinra (IL-1RA) (PeproTech) was injected IP, dissolved in 200 μL of PBS.
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