Transfected cells were washed twice with serum-free DMEM and stimulated with rat recombinant TNF-α (0.1 to 1ng/mL, Peprotech) for 17 h. The cells were then incubated with 250µM benzylamine at 37°C for 2 h. Since benzylamine is a substrate for both SSAO and MAO-B, the cells were pretreated with 0.5mM pargylin, a MAO-B inhibitor, for 30 min. SSAO activity in the supernatant was measured using a Fluoro SSAO kit (Cell technology).
Recombinant rat tnf α
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant rat TNF-α is a cytokine produced using recombinant DNA technology. It is a member of the tumor necrosis factor (TNF) superfamily and is involved in the regulation of immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Mk 150, by Takara Bio (1 mentions)
L6 cells, by American Type Culture Collection (1 mentions)
Epoxomicin, by Merck Group (1 mentions)
Z ietd fmk, by R&D Systems (1 mentions)
Nedd8, by Cell Signaling Technology (1 mentions)
Z vad fmk, by R&D Systems (1 mentions)
Procaspase 3, by Cell Signaling Technology (1 mentions)
Cullin3, by Cell Signaling Technology (1 mentions)
Pro caspase 7, by Cell Signaling Technology (1 mentions)
Phospho iκbα, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Necrostatin 1, by Merck Group (1 mentions)
Special culture medium, by Procell (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Dimethyl sulphoxide, by Merck Group (1 mentions)
Carboxymethylcellulose, by Merck Group (1 mentions)
Recombinant human tnf α, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit and antimouse igg hrp, by Bioworld Technology (1 mentions)
Collagenase type 2, by Merck Group (1 mentions)
10 protocols using recombinant rat tnf α
1
Measurement of SSAO Activity
2
Spermine-induced FDP-Lys Quantification
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Transfected cells were washed twice with serum-free DMEM and stimulated with rat recombinant TNF-α (0.1 to 1ng/mL, Peprotech) for 17 h. The cells were then incubated with 200µM spermine and 1mg/mL human serum albumin in PBS at 37°C. The level of FDP-Lys in the supernatant was determined using an ELISA kit (MK-150, Takara Bio).
3
Myotube Differentiation and Treatment
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Myoblasts derived from rat skeletal muscle (L6 cells; ATCC, Manassas, Virginia, USA) were cultured in 6-well plates containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin at 37°C under a humidified 5%CO2/95%O2 atmosphere. When myoblasts were approximately 75% confluent, myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 1% FBS. Differentiation was allowed to continue for 7 days before experimentation.
Fully differentiated L6 myotubes were treated and incubated for 24 h with recombinant rat TNFα (PeproTech, Princeton, New Jersey, USA) (10 μg/ml) and/or D-Trp(8)-γMSH (American Peptide, Sunnyvale, CA, USA) (0, 50 and 200 nM) or DMEM alone. At this concentration (10μg/ml) TNFα induces activation of NF-kB and down-regulation of IGF-I and Akt in C2C12 cells [26 (link)]. At the end of the incubation period, total RNA or proteins from cells were extracted.
Fully differentiated L6 myotubes were treated and incubated for 24 h with recombinant rat TNFα (PeproTech, Princeton, New Jersey, USA) (10 μg/ml) and/or D-Trp(8)-γMSH (American Peptide, Sunnyvale, CA, USA) (0, 50 and 200 nM) or DMEM alone. At this concentration (10μg/ml) TNFα induces activation of NF-kB and down-regulation of IGF-I and Akt in C2C12 cells [26 (link)]. At the end of the incubation period, total RNA or proteins from cells were extracted.
4
Rat TNF-α Stimulation of BMEC and SCMEC
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The day of the experiment, the inserts containing the BMEC and SCMEC monolayers were transferred to new six-well plates without astrocytes and stimulated for 12, 24, and 48 h with recombinant rat TNF-α 5 ng/mL (Peprotech, Rocky Hill, NJ, USA). Supernatants were collected, centrifuged, and stored at −80 °C until analysis. Rat CCL2 levels were evaluated using commercially available ELISA kits (Peprotech) according to the manufacturer’s instructions. All samples were analyzed in duplicates.
5
Pevonedistat Synthesis and Reagent Details
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Pevonedistat was synthesized by Millennium Pharmaceuticals, Inc. The following reagents were purchased from their respective companies: recombinant rat TNF-α (Peprotech, Rocky Hill, NJ, USA); caspase inhibitors Z-VAD-FMK and Z-IETD-FMK (R&D Systems, Minneapolis, MN, USA); Necrostatin-1 (Sigma-Aldrich, St. Louis, MO, USA); and Epoxomicin (Sigma-Aldrich). Antisera were purchased from the following companies: β-Actin, cleaved caspase-3, cleaved caspase-8 (p18), cFLIP, cullin-3, IκBα, phospho-IκBα, NEDD8, PARP, pro-caspase-3, pro-caspase-6, pro-caspase-7, pro-caspase-8 (p10), pro-caspase-9 (Cell Signaling, Danvers, MA, USA); MLKL (Millipore, Billerica, MA, USA); CDT1 (Santa Cruz Biotechnology, Dallas, TX, USA); and BID (eBioscience, San Diego, CA, USA). Complete antisera details are provided in Supplementary Information .
6
Baicalin Modulates Rat PASMC Response to TNF-α
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Rats PASMC were purchased from Procell Life Science & Technology Co, Ltd., and cultured in special culture medium (Procell) supplemented with 100 Ug/mL of penicillin, 100 IU/mL streptomycin, and 10% fetal bovine serum at 37°C in an incubator. Cells were passaged after 80% confluence, digested with 0.05% trypsin including 0.04% ethylene diamine tetraacetic acid (EDTA, Sigma‐Aldrich) in PBS. Recombinant rat TNF‐α was purchased from PeproTech (Cat:400‐14). Cells were treated with TNF‐α (5 ng/mL) for 24 h at 37°C in the presence of baicalin (100 μg/mL) for treatment or an equal volume of DMSO in culture medium group, respectively.
7
Polydatin Attenuates Osteoarthritis Progression
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Polydatin (purity > 98%) was purchased from Shanghai Aladdin Biological Technology Co, Ltd (Nantong, China). Dimethylsulphoxide (DMSO), carboxymethylcellulose (CMC) and type II collagenases were acquired from Sigma‐Aldrich (St Louis, MO). Recombinant rat TNF‐α was purchased from PeproTech (Rocky Hill, New Jersey, USA). The primary antibody against MCL‐1, cleaved caspase‐3, collagen II, aggrecan, MMP‐13, MMP‐3, ADAMTS‐4, Nrf2, HO‐1, Keap1 and Alexa Fluor®488‐labelled goat antirabbit IgG (H+L) second antibody and Alexa Fluor®594 goat antimouse IgG (H+L) second antibody were purchased from Abcam (Cambridge, UK). p16 and p53 antibodies were purchased from Sigma‐Aldrich. Antibody LC3 purchased from Cell Signaling Technology (Danvers, MA) Recombinant human TNF‐α was obtained from PeproTech and goat antirabbit, and antimouse IgG‐HRP were from Bioworld (Minneapolis, Minnesota, USA). The 4′, 6‐diamidino‐2‐phenylindole (DAPI) was purchased from Beyotime (Shanghai, China). Cell culture reagents were obtained from Gibco (Grand Island, NY).
8
Permeability Assay for Barrier Integrity
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After 24 h inflammation with recombinant rat TNF-α (5 ng/mL, Peprotech), barrier integrity of the in vitro models was controlled with Lucifer Yellow (LY CH lithium salt, Sigma Aldrich), a small hydrophilic molecule (MW 457 g/mol) retained by the monolayers as previously described [12 ]. Briefly, quantification of the LY paracellular leakage from the luminal to the abluminal compartment was assessed by fluorimetric analysis (excitation at 430 nm and emission at 535 nm) and expressed in LY permeability, Pe(LY). Barrier integrity was validated for Pe(LY) below 0.6.10−3 cm/min.
9
ADSC Inflammatory Response to TNF-α
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ADSCs were treated with recombinant rat TNFα (Peprotech USA) at the concentration of 10 ng mL−1 for 16-18 h. In brief, ADSCs were plated in six-well plates (Corning) at the density of 2 × 105 cells per well. After incubation with DMEM F12 containing 10% heat-inactivated FBS for 24 h, the medium was changed to serum-free DMEM F12 containing 10 ng mL−1 TNF-α (Peprotech). After incubation for 18 h, the cells were harvested and used for further analyses.
10
Daphnetin and Triptolide Pathway Investigation
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Daphnetin (DAP) standard, purity ≥ 98%, was purchased from Shanghai Source Leaf Biological Technology Co., Ltd. Triptolide (TP) standard, purity ≥ 98%, was purchased from Chengdu Pfeiffer Biotechnology Co., Ltd. Recombinant Rat TNF-α was purchased from PeproTech. NT-CROZ, anti-p38, and anti-JNK were purchased from the United States of America Abcam Corporation. Anti-phospho-p38, antiphospho-p38, anti-phospho-p38, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP were purchased from the United States KPL company. All primers were designed by the BGI gene software and tested by NCBI BLAST.
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