Costunolide (purity >98%; Fig. 1A ) was obtained from Nantong Jingwei Fine Chemical Co., Ltd., and was dissolved in dimethyl sulfoxide (DMSO). Rat IL-1β was purchased from R&D Systems, Inc. Chloral hydrate and DMSO were purchased from Sigma-Aldrich (Merck KGaA). FBS, Dulbecco's modified Eagle's medium (DMEM), streptomycin, penicillin, 0.25% trypsin and collagenase II were obtained from Gibco (Thermo Fisher Scientific, Inc.).
Rat il 1β
Manufactured by R&D Systems
Sourced in United States
Rat IL-1β is a recombinant protein that represents the mature form of the rat interleukin-1 beta cytokine. Interleukin-1 beta is a pro-inflammatory cytokine involved in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Chloral hydrate, by Merck Group (1 mentions)
Collagenase 2, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Total ros detection kit, by Enzo Life Sciences (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rat insulin elisa kit, by ALPCO (1 mentions)
Apoptotic dna ladder detection kit, by Thermo Fisher Scientific (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Enzyme linked immunosorbent assay elisa kit, by R&D Systems (1 mentions)
Rat tnf α, by R&D Systems (1 mentions)
Hplc grade ethanol, by Merck Group (1 mentions)
Annexin 5 fitc propidium iodide kit, by Thermo Fisher Scientific (1 mentions)
Aspirin enteric coated tablets, by Bayer (1 mentions)
Adi 900 097, by Enzo Life Sciences (1 mentions)
Tnf α, by BD (1 mentions)
Recombinant human tgf β1, by BioLegend (1 mentions)
8 protocols using rat il 1β
1
Costunolide Extraction and Preparation Protocol
2
Expression and Purification of PEP-1-PON1 Fusion Protein
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The cell-permeable fusion protein PEP-1-PON1 was expressed and purified as previously described (26 (link)). A schematic representation of the expression vector for PEP-1-PON1 and the expressed fusion protein is shown in Fig. 1A . Rat IL-1β, TNF-α, and IFN-γ were obtained from R & D Systems (Minneapolis, MN, USA), Enzo (Farmingdale, NY, USA), and Chemicon (Billerica, MA, USA), respectively. Total ROS detection kit was obtained from Enzo (Plymouth Meeting, PA, USA). Rat insulin ELISA kit was purchased from Alpco (Salem, NH, USA). Alexa Fluor 488 secondary antibody and apoptotic DNA ladder detection kit were obtained from Invitrogen (Frederick, MD, USA). Most of the antibodies used in western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA).
3
Aspirin's Anti-inflammatory Mechanisms
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HPLC-grade ethanol was purchased from Merck Company (Darmstadt, Germany). Analytical grade acetic acid and ultrapure water (Watsons, Guangzhou, China) were used throughout the experiment. Instant dry yeast was obtained from Angel Yeast Co., Ltd. (Yichang, China). Aspirin enteric-coated tablets were supplied by Bayer S.p.A. (Viale Certosa, Milano, Italy). The cell counting kit 8 (CCK-8) was purchased from Nanjing Sunshine Biotechnology Co., Ltd. (Nanjing, China). The Annexin V-FITC/propidium iodide (PI) kit was supplied by Invitrogen (California, USA). Enzyme-linked Immunosorbent Assay (ELISA) Kits, Rat TNF-α, and Rat IL-1β were supplied by R&D Systems (Minnesota, USA). ELISA Kits for rat endothelin-1 (ET-1), rat intercellular adhesion molecule-1 (ICAM-1), rat matrix metallopeptidase 9 (MMP-9), rat NF-κB, rat nitric oxide (NO), rat thromboxane B2 (TXB2), and rat superoxide dismutase (SOD) were procured from Nanjing Senbeijia Biological Technology Co., Ltd. (Nanjing, China).
4
Plasma Corticosterone and IL-1β ELISA
5
Cytokine quantification by ELISA
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ELISA was employed to detect Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, and interleukin-6, IL-6, and IL-10 concentrations using a rat IL-1β, TNF-α, IL-6, and IL-10 ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) following the manufacturer's instruction.
6
ELISA Assay for Rat Cytokines
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7
Th17 Differentiation from Rat CD4+ T-cells
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CD4+ T-cells from male Lewis rats were obtained from splenocytes and used in Th17 differentiation assays as previously described [23 (link)]. In brief, cells were incubated with the following rat-specific antibodies: CD8a (clone OX8; BioLegend), CD45RA (clone OX33; BioLegend), CD11b/c (clone OX42; BD Biosciences), CD25 (clone OX39; eBioscience), and erythrocytes (clone OX83; Cedarlane), followed by a washing step. Cells were incubated with anti-IgG coated microbeads (Miltenyi Biotec) and unwanted cells were magnetically depleted using a MACS column system according to the manufacturer`s instructions (Miltenyi Biotec). CD4+ T-cells were stimulated with rat-specific anti-CD3 (pre-coated to the plated at 4 μg/ml, clone G4.18) and soluble anti-CD28 antibody (2 μg/ml, clone JJ319, BioXcell) together with recombinant human TGF-β1 (5 ng/ml, BioLegend), mouse IL-6 (20 ng/ml, BD Pharmingen), rat IL-1β (10 ng/ml, R&D Systems), and mouse IL-23 (10 ng/ml, BioLegend) in the presence of anti-rat IFN-γ antibody (5 μg/ml, BioLegend). After 72 hrs of incubation, the supernatants were harvested and IL-17A cytokine concentrations were quantified by ELISA according to the manufacturer`s specifications (eBioscience).
8
Sandwich ELISA for Cytokine Quantification
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Cytokine concentration in cell culture supernatants was determined by sandwich ELISA using MaxiSorp plates (Nunc, Rochild, Denmark) and anti-cytokine paired antibodies according to the manufacturer's instructions. Samples were analyzed in duplicates for rat IL-1β, IL-6, IL-10 (R&DSystems, Minneapolis, MN), TNF (eBioscience), IL-12, p40 and transforming growth factor (TGF- (BioSource, Camarrilo, CA). The results were calculated using standard curves made on the basis of known concentrations of the appropriate recombinant cytokines.
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