Monocytes were isolated by immunomagnetic cell sorting (MACS Cell Isolation kits; Miltenyi Biotec, Bergisch Gladbach, Germany). Positive selected CD14+ cells were analyzed by flow cytometry. Purity of the CD14+ cells was >98%. Purified CD14+ monocytes were cultured in RPMI-1640 medium containing 10% FBS, 100 U/ml penicillin and 100 ng/ml streptomycin at the concentration of 1×106/ml, supplemented with 1,000 U/ml IFN-α2b (Anterferon;, Anhui, China) and 40 ng/ml GM-CSF for IFN-DC or 20 ng/ml IL-4 (both from R&D Systems, Minneapolis, MN, USA) and 40 ng/ml GM-CSF for IL-4-DC. The cells were incubated at 37°C and 5% CO2 for 5 days. Half of the supernatants were moved and fresh cytokines and mediums were added every 3 days. DCs were matured by adding 20 ng/ml tumor necrosis factor-α (TNF-α; R&D Systems) and culturing for another 48 h.
Tumor necrosis factor α tnf α
Manufactured by R&D Systems
Sourced in United States
Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine. It plays a central role in regulating immune responses and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Lipofectamine reagent, by Promega (3 mentions)
Penicillin, by Corning (3 mentions)
Kallikrein, by Enzyme Research (3 mentions)
Interleukin 4 (il 4), by R&D Systems (2 mentions)
Interferon γ ifnγ, by R&D Systems (2 mentions)
Dibucaine, by Merck Group (2 mentions)
Caspase 3 inhibitor, by Merck Group (2 mentions)
Pd98059, by Merck Group (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Interleukin 1β il 1β, by R&D Systems (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Macs cell isolation kit, by Miltenyi Biotec (1 mentions)
Ang 2, by R&D Systems (1 mentions)
Mouse mfg e8, by R&D Systems (1 mentions)
Haosmc, by Lonza (1 mentions)
Smgm 2 medium, by Lonza (1 mentions)
Human mfg e8, by R&D Systems (1 mentions)
Collagenase 2, by Worthington (1 mentions)
30 protocols using tumor necrosis factor α tnf α
1
Generation of IFN-DCs and IL-4-DCs from Monocytes
2
Isolation and Characterization of Mouse Vascular Smooth Muscle Cells
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Mouse VSMCs were enzymatically isolated as described in the literature [33 (link)]. In brief, the thoracic aortas of the young and aged mice were isolated, and the adventitia and intima were removed from the vessels. The aortae were cut into 1–2-mm pieces and placed into tubes containing 2 mg/mL collagenase II (Worthington Biochemical, Lakewood, NJ, USA) at 37 °C for 3–5 h. The isolated cells were washed with and plated in complete Dulbecco’s Modified Eagle’s Medium (DMEM). To ensure the purity of the isolated cells, > 95% of the cells had to be positive for the two specific smooth muscle cell markers: smooth muscle α-actin and smooth muscle myosin heavy chain for them to be used [34 ]. Early passage VSMCs were treated with Ang II (1 μM, R&D Systems), tumor necrosis factor-α (TNF-α, 20 ng/mL, R&D Systems), or mouse MFG-E8 (250 ng/mL, R&D Systems) for 24 h prior to the subsequent experiments. Human aortic smooth muscle cells (hAoSMCs) were purchased from Lonza (Basel, Switzerland) and cultured in SmGM-2 medium (Lonza) for 24 h in either the presence or absence of human MFG-E8 (250 ng/mL, R&D systems).
3
Platelet Activation Assay Protocol
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Soluble collagen was from Corning (Corning, NY, USA). Prostaglandin I2 (PGI2) was from Cayman Chemical (Ann Arbor, MI, USA). Crosslinked collagen-related peptide (CRP-XL) was from R. Farndale (CambCol Laboratories, Cambridge University, UK). CHRONO-LUME® detection reagent was from Chrono-Log Corporation (Havertown, PA, USA; #395). Pam2CSK4 and Pam3CSK4 were from Invivogen (#tlrl-pm2s-1 and #tlrl-pms). Tumor necrosis factor-α (TNF-α, #210-TA-020) was from R&D Systems. FSL-1 was from Tocris (#6011). Anti-TLR2 (#maba2-htlr2), anti-TLR6 (magb-htlr6) and control IgG (mabg1-ctrlm) and IgA (maba2-ctrl) antibodies were from Invivogen. All other reagents were from Sigma-Aldrich or previously named sources (48 (link)).
4
Cellular Assays for Oxidative Stress
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M199 medium, gelatin, penicillin, streptomycin, heparin, NaOH, sodium dodecylsulfate (SDS), Tris, phosphate-buffered saline (PBS), ethylendiaminetetraacetic acid (EDTA), chloroform, glycerol, trichloroacetic acid, trypsin, hydroxyurea, glucose, bromophenol blue, lipofectamine, CO-releasing molecule-2 (CORM2), NADPH, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, Triton X-100, and mercaptoethanol were from Sigma-Aldrich (St. Louis, MO, USA). Bilirubin and tin protoporphyrin were from Frontier Scientific (Logan, UT, USA). The antibody against HO-1 was from Assay Designs (Ann Arbor, MI, USA) and the antibody against β-actin was from Santa Cruz (Santa Cruz, CA, USA). Tumor necrosis factor-α (TNFα) was from R&D Systems (Minneapolis, MN, USA). Canagliflozin was purchased from Selleck Chemicals (Houston, TX, USA). [3H]Thymidine was from Perkin Elmer (Boston, MA, USA).
5
Procuring Reagents for TNFα Assay
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S1P (Biomol) was purchased through Enzo Life Sciences (Exeter, UK) and tumor necrosis factor α (TNFα) purchased from R&D Systems (Minneapolis, MN). Unless otherwise specified, all chemicals were from Sigma-Aldrich (St. Louis, MO).
6
Immune Cell Phenotyping and Cytokine Profiling
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Cell staining was performed using fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), or pacific blue (PB)-conjugated mouse monoclonal antibodies against CD80, CD86, CD83, CD14, and CD163 (all purchased from BD Biosciences, San Jose, CA, USA). Fluorochrome conjugated antibodies against CD8, CD4, CD56, and CD19 were purchased from BioLegends (Nordic BioSite AS, Kristiansand, Norway). The following cytokines were used: Interleukin-4 (IL-4), granulocyte-colony stimulating factor (GM-CSF), monocyte-colony stimulating factor (M-CSF), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and interferon-γ (IFN-γ; all purchased from R&D Systems (Minneapolis, MN, USA).
7
Hypoxia-Induced Cellular Responses
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For drug treatments, UFP-512, a specific and effective DOR agonist, was synthesized by our research teams (Balboni et al., 2002 (link); Chao et al., 2007 (link)), naltrindole, a particular DOR antagonist (Zhang et al., 2006 (link); Granier et al., 2012 (link)), was purchased from Sigma (St. Louis, MO, United States), tumor necrosis factor-α (TNF-α) was purchased from R&D Systems (Minneapolis, MN, United States), they were added into the culture medium in demanded concentrations and exposed to normoxic or hypoxic conditions starting immediately before the onset of hypoxia (or at the same time-point in normoxia).
8
TCDD-Induced Cellular Effects Evaluation
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TCDD (>99.99% purity; Dow Chemicals Co., Midland, MI, USA) was dissolved in dimethylsulfoxide (DMSO) and stored in the dark at −20°C until use. α-minimum essential medium (α-MEM; with glutamine) and antibiotics [penicillin (10,000 U/ml) and streptomycin (10,000 µg/ml); P/S] were purchased from Gibco Life Technologies Corp. (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Omega Scientific Inc. (Tarzana, CA, USA). 2-Methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH223191) was obtained from Selleckchem Co. (Houston, TX, USA) and was dissolved in DMSO. Tumor necrosis factor-α (TNF-α) was obtained from R&D Systems (Minneapolis, MN, USA) and gemcytabine from Hospira, Inc. (Lake Forest, IL, USA) and were diluted in phosphate-buffered saline (PBS). Caspase-3 inhibitor (CAS 169332-60-9-Calbiochem), crystal violet and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
9
Induction of iNOS Expression
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To induce iNOS expression, subconfluent monolayers were cultured in serum-free medium for 24 h. Growth-arrested cultures were treated with pro-inflammatory cytokines, 100 U/ml interferon γ (IFN-γ) (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/ml interleukin-1 α (IL-1α) (PeproTech, Inc., Rocky Hill, NJ, USA) and 25 ng/ml tumor necrosis factor-α (TNF-α) (R&D Systems, Minneapolis, MN, USA), pro-inflammatory cytokines and 0.1–5 mg/ml water extract of Cnidii Rhizoma or 0.5 mM 1400W (Sigma-Aldrich) in fresh medium without fetal bovine serum. After 48 h, the supernatants were collected and the cells were harvested and lysed as previously described (18 (link)).
10
Thrombin and Fibrinolysis Regulation Assays
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