The concentrations of IL-6, TNF, and PGE2 in the cell supernatants were measured using commercial human IL-6, TNF (R&D Systems), and PGE2 ELISA kits (R&D Systems), following the manufacturer’s protocol. As an indicator of NO production, nitrite was measured in the culture medium using the Griess Reagent System (Promega, Madison, WI, USA), following the manufacturer’s protocol. Optical density at 450 nm was determined using an Epoch microplate spectrophotometer (BioTek Instruments).
Pge2 elisa kit
Manufactured by R&D Systems
Sourced in United States
The PGE2 ELISA kit is a quantitative sandwich enzyme immunoassay designed for the measurement of PGE2 (Prostaglandin E2) levels in biological samples. The kit utilizes a specific antibody coated on a microplate to capture PGE2 from the samples. The captured PGE2 is then detected using a detection antibody conjugated to an enzyme, which catalyzes a color change reaction that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human il 1β, by R&D Systems (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Cytiva (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
Berberine, by Merck Group (2 mentions)
N 1 naphthyl ethylenediamine dihydrochloride, by Merck Group (2 mentions)
Penicillin streptomycin solution, by Welgene (2 mentions)
Phosphoric acid, by Merck Group (2 mentions)
Sulfanilamide, by Merck Group (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Tumor necrosis factor (tnf), by R&D Systems (1 mentions)
Griess reagent system, by Promega (1 mentions)
Mmp 3, by R&D Systems (1 mentions)
Mmp 13, by R&D Systems (1 mentions)
Mmp 1, by R&D Systems (1 mentions)
No detection kit, by Nanjing Jiancheng (1 mentions)
36 protocols using pge2 elisa kit
1
Quantification of Inflammatory Mediators
2
PGE2 Production in LPS-Stimulated RAW 264.7 Cells
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RAW 264.7 cells were treated with compounds (50 μM) 1 h prior to LPS (100 ng/mL) stimulation for 24 h. Cells were pretreated with positive controls (NS398) for 1 h, and then stimulated with LPS (100 ng/mL) for 24 h. The supernatant was collected and PGE2 production detected by using PGE2 ELISA Kits (R&D Systems, MN, USA).
3
Investigating LXRα and NF-κB Regulation
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SSa (purity>98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Geranyl geranyl pyrophosphate (GGPP) was purchased from Sigma Chem. (St. Louis, USA). Recombinant human IL-1β, MMP1, MMP3, MMP13, and PGE2 ELISA kits were purchased from R&D systems (Minneapolis, MN, USA). Antibodies for LXRα, IκBα, and NF-κB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NO detection kit was purchased from Nanjing Jiancheng Bioengineering Institute. (Nanjing, China).
4
Measurement of Nitric Oxide and PGE2 in Macrophages
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As described in Section 4.4 ., macrophages were stimulated with LPS (1 µg/mL) only or in combination with P4P (12.5, 25, or 50 µM) and further incubated another 24 h. The cultured cell supernatant (100 μL) was mixed with an equal volume of Griess reagent and incubated at room temperature for 10 min, and absorbance was measured at 540 nm to measure NO production [64 (link),65 (link)]. PGE2 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). In each case, sample measurements were performed in duplicate. The optical density was determined on a microplate spectrophotometer using a 450 nm wavelength.
5
Cytokine and Metabolite Profiling in LPS-Treated Cells
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Cells were harvested and centrifuged to collected cultured media at time points after LPS (Millipore) treatment. Cytokine and metabolite concentrations were measured by ELISA. IL-6, IL-10 and PGE2 ELISA kits purchased from R&D Systems. ELISA assays were performed according to the manufacturer’s instructions.
Cytokine antibody array: Mouse brains were homogenized in 500 μL of cold PBS containing protease inhibitor cocktail and 5 μL of Triton-X100 and centrifuged at 10,000×g for 5 min at 4 °C. Obtained lysates (300 μg) were subjected to cytokine protein array. Cytokine expressions were detected by a mouse cytokine antibody array, panel A kit according to the manufacturer’s instructions. Obtained spots were measured as densities by ImageJ software and showed in a graph. Cytokine mouse antibody array were performed according to the manufacturer’s instructions.
Cytokine antibody array: Mouse brains were homogenized in 500 μL of cold PBS containing protease inhibitor cocktail and 5 μL of Triton-X100 and centrifuged at 10,000×g for 5 min at 4 °C. Obtained lysates (300 μg) were subjected to cytokine protein array. Cytokine expressions were detected by a mouse cytokine antibody array, panel A kit according to the manufacturer’s instructions. Obtained spots were measured as densities by ImageJ software and showed in a graph. Cytokine mouse antibody array were performed according to the manufacturer’s instructions.
6
Murine Mast Cell Signaling Pathway
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Murine MCs were kindly provided by donors Fudan University; cAMP and PGE2 ELISA kits were obtained from R&D; TGF-β1 was obtained from Peprotech; 17-phenyl PGE2 (EP1A, EP1 agonist, prostanoid receptor agonist selectivity: EP1>EP3), butaprost (EP2A, EP2 agonist), sulprostone (EP3A, prostanoid receptor agonist selectivity: EP3>EP1) and cay10580 (EP4A, EP4 agonist) were obtained from Cayman; Rabbit anti-phospho-p38 MAPK (Thr-180/Tyr-182), phospho-ERK1/2 (Thr-202/Tyr-204), phospho-JNK1/2 (T183/Y185), total JNK, total ERK1/2, total p38, p27kip1, cyclin D1, COX-2 were obtained from Cell Signaling Technology. Mouse anti-CCN2/connective tissue growth factor (CTGF), membrane-bound PGE synthase 1 (mPGES1), laminin (LN) were obtained from Abcam; Dulbecco's modified Eagle's medium, phosphate-buffered saline, trypsin, non-essential amino acids and antibiotics (penicillin/streptomycin) were obtained from Invitrogen; all solutions and instruments for RT-PCR were obtained from Applied Biosystems; 100-mm and 6-well cell culture plates were obtained from Fisher. The experimental protocol was approved by the Experimentation Ethics Committee of the Nantong University for the use of human or animal subjects.
7
Characterizing Anti-Inflammatory Mechanisms of Novel Lipid Mediator
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PCTR1 (17-hydroxydocosa-4,7,10,12,14,19-hexaenoic acid) was obtained from Cayman Chemical Company (Ann Arbor, MI). BOC-2 (ALX antagonist) was purchased from Biomol-Enzo Life Sciences (Farmingdale, NY). Interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor-α (TNF-α), macrophage inflammatory protein 2 (MIP-2), myeloperoxidase (MPO), and PGE2 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Serum glutamic pyruvic transaminase (ALT), serum glutamic oxaloacetic transaminase (AST), blood urea nitrogen (BUN), blood creatinine (CREA), reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathioneperoxidase 4 (GPX4) kits were purchased from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). PLA2 ELISA kits were purchased from WESTTANG BIO-TECH CO. (Shanghai, China). High performance liquid chromatography-mass spectrometry (HPLC-MS) kits for LA and AA were purchased from Dr. Ehrenstorfer (Augsburg, Germany). LPS (Escherichia coli serotype 055: B5) was obtained from Sigma (St. Louis, MO). Quantitative real-time PCR kits were purchased from TaKaRa (Japan).
8
PGE2 Quantification by ELISA
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The concentration of PGE2 in the culture supernatant was determined by using a PGE2 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.
9
Evaluation of Anti-inflammatory Effects
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Cells were seeded at 1 × 105 cells per well in 96-well plates and pre-treated with various concentration of FF-8 for 2 hr followed by treatment with LPS (500 ng/mL) for 24 hr in the presence of FF-8. Cell culture supernatants were collected and mixed with an equal volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid, and 0.1% naphthylethylenediamide) at room temperature for 10 min. The absorbance was then read at 540 nm using a microplate reader. NO concentrations in the supernatant were determined by comparison with a standard curve. The level of PGE2 in cell culture media was measured using the PGE2 ELISA kit according to the manufacturer's introductions (R&D Systems, Minneapolis, MN, USA). Cell viability was measured after 24 hr of exposure to FF-8 based on the ability of mitochondria in viable cells to reduce 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Co., St. Louis, MO, USA) [18 (link)].
10
Inflammatory Cytokine Assay Protocol
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The chemicals included methanol, dimethyl sulfoxide (DMSO), and sodium chloride purchased from Geochim Sarl, West Region of Cameroon. Dextran sodium sulfate was purchased from Tokyo Chemical Industry Co., LTD, Japan. Luminol, lymphocyte separation medium, lucigenin, and Hank's balanced salt solution were obtained from MP Biomedicals Inc., Research Organics, and Sigma. Phorbol myristate acetate and zymosan A were purchased from Fluka. Human monocytic leukemia cells were gotten from European Collection of Cell Cultures. Ammonium chloride of analytical grades and ethanol were from Merck Chemicals, Darmstadt, Germany. Human TNF-α, IL-1β, IL-6, and PGE2 ELISA Kit were from R&D Systems, Minneapolis, USA. Glass fiber filter and cell harvester were from Inotech, Dottikon, Switzerland. Prednisolone (Solupred) and pediatric catheter were purchased from local pharmacy.
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