RAW 264.7 mouse monocytic cells were purchased from ATCC. Cells were treated with 100ng/ml LPS from Escherichia coli serotype 0111:B4 (Sigma), 10^-6M MCH (Bachem), 10 ng/ml IL-10 (R&D Systems), 10ng/ml IL-1beta (R&D Systems), their various combinations or vehicle, and were harvested at 4 hours post-treatment for RNA analysis and at 72 hours for protein secretion in the tissue culture supernatant. Experiments using RAW cells were run in 5–6 replicas. IL-10 levels in culture supernatant were measured by ELISA (R&D systems). RNA was extracted using RNAqueous extraction kit (Ambion). Reverse transcription was performed using an I-Script cDNA synthesis kit (Bio-Rad Laboratories), followed by real-time PCR analysis of IL-10 or MCHR1 expression using Taqman pre-developed assays (Applied Biosystems). TATA-binding protein (TBP) gene expression served as a housekeeping control and results are expressed as arbitrary mRNA units (AU) (vehicle=100), unless indicated otherwise.
Il 1beta
Manufactured by R&D Systems
Sourced in United States
IL-1beta is a recombinant human protein that belongs to the interleukin-1 family. It is a key mediator of the inflammatory response and is involved in a variety of cellular activities.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by R&D Systems (2 mentions)
Lps from escherichia coli serotype 0111 b4, by Merck Group (1 mentions)
Rnaqueous extraction kit, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Elisa, by R&D Systems (1 mentions)
Il 10, by R&D Systems (1 mentions)
Il 13, by Abcam (1 mentions)
Cxcl10, by Thermo Fisher Scientific (1 mentions)
Il 1alpha, by R&D Systems (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Glutamine, by Merck Group (1 mentions)
Penicillin streptomycin, by Euroclone (1 mentions)
Rpmi 1640 medium, by Euroclone (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Interferon gamma, by R&D Systems (1 mentions)
Gapdh 14c10, by Cell Signaling Technology (1 mentions)
Irdye 680cw, by LI COR (1 mentions)
Rosiglitazone, by Cayman Chemical (1 mentions)
P hsl s563, by Cell Signaling Technology (1 mentions)
Beta actin 8h10d10, by Cell Signaling Technology (1 mentions)
6 protocols using il 1beta
1
Regulation of IL-10 and MCHR1 in RAW 264.7 Cells
2
Cytokine Profiling in Paw Tissue
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Paw tissue was retrieved at 96 h post CFA injection and minced in ice cold lysis buffer (20 mM imidazole hydrochloride, pH 6.8; 100 mM potassium chloride, 1 mM magnesium chloride, 10 mM ethylene glycol tetraacetic acid, 1.0% Triton X-100, 10 mM sodium fluoride, 1 mM sodium molybdate, 1 mM ethylenediaminetetraacetic acid (Sigma-Aldrich, Munich; Merck, Darmstadt; Carl Roth GmbH, Karlsruhe, all Germany) with complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany). The homogenate was frozen at −80°C. Before experiment, the homogenate was thawed, incubated at 4°C overnight, centrifuged at 14,000 g for 10 min and the supernatant was used for ELISA [13] (link). ELISA kits were used according to the manufacturer's instructions: IL-1alpha, IL-1beta and interferon (IFN)-gamma (R&D systems, London, UK); CXCL10 (Peprotech, Hamburg, Germany); TNF-alpha and IL-4 (Invitrogen, Life Technologies, Darmstadt, Germany) and IL-13 (Abcam, Cambridge, UK).
3
Serum Cytokine Quantification by ELISA
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Serum cytokine levels were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s specifications (Table 1 ). Kits for IL-6 (reference values: 0.447–9.96 pg/ml, minimal detectable level: 0.016–0.110 pg/ml), and IL-1 beta (reference values: <1.996 pg/ml, minimal detectable level: <0.1 pg/ml) were purchased from R&D Systems (Minneapolis, USA). For TNF alpha (DPC Biermann GmbH, Bad Nauheim, Germany), the reference values from the manufacturer were <8.1 pg/ml and minimal detectable level was 0.1 pg/ml. Intra-assay precision was 2.6–3.6%, and inter-assay precision was 4–6.5%.
4
Activation of SLN Cells by Inflammatory Cytokines
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SLN cells were re-suspended in RPMI 1640 medium (EuroClone) supplemented with 1% penicillin/streptomycin (EuroClone) and 2% glutamine (Sigma), without serum, indicate as culture medium. For in vitro stimulation analyses, cells from each SLN were divided into three aliquots: the first was processed immediately, indicated as t = 0; the second and the third were cultured for 24 h in culture medium at 37 °C and 5% CO2 with or without the following inflammatory cytokines: TNF-alpha (10 ng/mL), IL-1beta (10 ng/mL) and IL-6 (1000 U/mL), all from R&D Systems (Minneapolis, MN). Cells were used for fluorescent immunocytochemistry and flow cytometry.
5
Human and Murine Adipocyte Experiments
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Collagenase type II and isoproterenol used in human adipocyte experiments, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and PAR1 (TFFLR) and PAR2 (SLIGRL) agonists used in 3T3-L1 adipocyte experiments were all from Sigma-Aldrich Sweden AB. TNF-alfa, interferon-gamma, and IL-1beta were from R&D Systems, USA. insulin used in human adipocyte experiments was from Novo Nordisk A/S, Denmark. Human recombinant active site-inhibited FVIIa (FVIIai) and recombinant human and murine FVIIa were kind gifts from L-C Petersen, Novo Nordisk. FX was from Enzyme Research Lab, USA. Rosiglitazone was from Cayman Chemical, USA. TaqMan probes were from Applied Biosystems, USA. The following antibodies were used: GAPDH 14C10, Beta actin 8H10D10, Cleaved caspase-3 D175, pHSL s563, and HSL from Cell Signaling Technology, USA; TF N1C3 from GeneTex USA; TF AF2339 and TF AF3178 from R&D Systems, USA; IRDye 680CW and IRDye 800CW from LI-COR Biosciences, UK.
6
Immunoprecipitation of FLAG-tagged Syndecan-4
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Immunoprecipitation was performed using Anti-FLAG M2 Magnetic Beads (Sigma) according to the manufacturer's protocol. Therefore, we transfected HEK cells either with FLAG tagged full length sdc4 (flag-sdc4wt) or with a FLAG-tagged mutant of sdc4 in which all GAG binding serine residues were replaced by alanine (flag-sdc4S3A) and that therefore has no GAG side chains.
The next day, cells were incubated with 100 ng IL-1beta or TNF alpha (both R&D) for 1 h at 37 °C in DMEM medium without FCS. IL-1 was detected using an IL-1beta Antibody (#12242, Cell signaling) and TNF using a TNF alpha antibody (#3707, Cell signaling). True Blot secondary antibodies (Rockland) were used to ensure, that IgG fragments from the antibodies attached to the beads were not detected.
The next day, cells were incubated with 100 ng IL-1beta or TNF alpha (both R&D) for 1 h at 37 °C in DMEM medium without FCS. IL-1 was detected using an IL-1beta Antibody (#12242, Cell signaling) and TNF using a TNF alpha antibody (#3707, Cell signaling). True Blot secondary antibodies (Rockland) were used to ensure, that IgG fragments from the antibodies attached to the beads were not detected.
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