The mouse macrophage cell line RAW264.7 was purchased from ATCC and incubated in RPMI 1,640 with 10% fetal bovine plasma in a 37°C sterile incubator with 5% CO2. Cells were seeded in 6- or 24 -well plates and exposed to TPPU (1 μM, Cayman Chemical, Ann Arbor, Mich) or 14,15-EET (1 μM, Cayman Chemical, Ann Arbor, Mich) for 4 h after they reached 80% to 90% confluence. LPS (1 μg/mL) was then added and co-incubated for 12 h. 14,15-EEZE (an inhibitor of 14,15-EET, 1 μM, Cayman Chemical, Ann Arbor, Mich) was added 1 h before LPS stimulation. Cell supernatants were collected for inflammatory cytokines detection (20 (link)).
14 15 eet
Manufactured by Cayman Chemical
Sourced in United States
14,15-EET is a chemical compound used in research applications. It is a metabolite of arachidonic acid and belongs to the epoxyeicosatrienoic acid (EET) family of lipid signaling molecules. 14,15-EET has been studied for its potential biological functions, but a detailed description of its core function is not available while maintaining an unbiased and factual approach.
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Lab products found in correlation
11 12 eet, by Cayman Chemical (9 mentions)
8 9 eet, by Cayman Chemical (6 mentions)
5 6 eet, by Cayman Chemical (4 mentions)
14 15 eeze, by Cayman Chemical (3 mentions)
Matrigel, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Thiazolyl blue tetrazolium bromide, by Merck Group (1 mentions)
Antibody for β actin, by Merck Group (1 mentions)
Cay10441, by Cayman Chemical (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Ho 1 antibody, by Enzo Life Sciences (1 mentions)
Pge2 enzyme linked immunosorbent assay elisa kit, by Enzo Life Sciences (1 mentions)
Lamin a c antibody, by Cell Signaling Technology (1 mentions)
Sulforhodamine b based in vitro toxicology assay kit, by Merck Group (1 mentions)
Cox 2, by Santa Cruz Biotechnology (1 mentions)
Ah6809, by Cayman Chemical (1 mentions)
L 161 982, by Cayman Chemical (1 mentions)
Collagenase, by Worthington (1 mentions)
Transwell permeable support, by Corning (1 mentions)
17 protocols using 14 15 eet
1
Macrophage Modulation by EET and TPPU
2
Wound Healing Promotion with EET Derivatives
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Wounds were created on the dorsum of both ears three days after the induction of ischemia. To attain a standard size of the wounds a circular punch (diameter 2.25 mm) was used to incise a full thickness layer of ear skin down to the underlying cartilage, as described previously. After the punch incision, the skin was carefully removed from the underlying cartilage. Subsequently, the wound was covered with a small methylcellulose pad according to the treatment group with or without 11,12 EET or 14,15 EET (Cayman Chemical Company, Ann Arbor, Michigan, USA). Pads were manufactured by taking 200μl of 2,5% carboxymethylcellulose in PBS and adding either 200μl ethanol for control or 160μl ethanol and 40μl of 95% 11,12 EET or 98% 14,15 EET to the mixture. To obtain a pad the mixture was dried for 20 minutes on a hot plate at 37° Celsius on parafilm.
Finally, the entire ear was covered with a bioadhesive dressing (Opsite; Smith and Nephew Medical Ltd., Tuttlingen, Germany) [24 ,22 (link)].
Finally, the entire ear was covered with a bioadhesive dressing (Opsite; Smith and Nephew Medical Ltd., Tuttlingen, Germany) [24 ,22 (link)].
3
Evaluation of Anti-Inflammatory Compounds
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12-(((tricyclo(3.3.1.13,7)dec-1-ylamino)carbonyl)amino)-dodecanoic acid (AUDA), 8,9-EET, 11,12-EET, 14,15-EET, CAY10441, L-161,982, and AH6809 were purchased from Cayman Chemical (Ann Arbor, MI, USA). 1-Cyclohexyl-3′-dodecylurea (CDU) and ICI-192,605 were obtained from Calbiochem (Darmstadt, Germany). PDGF was supplied by Peprotech (Rocky Hill, NJ, USA). Antibodies for Pin1, Keap1, Nrf2, COX-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HO-1 antibody and PGE2 enzyme-linked immunosorbent assay (ELISA) kit were obtained from Enzo Life Sciences (Ann Arbor, MI, USA), and Lamin A/C antibody was from Cell Signaling Technology (Beverly, MA, USA). Antibody for β–actin, thiazolyl blue tetrazolium bromide (MTT), elastase, Sulforhodamine B based in vitro toxicology assay kit, hematoxylin and eosin solution were supplied by Sigma-Aldrich (St. Louis, MO, USA). Collagenase was purchased from Worthington Biochemical Corporation (Lakewood, NJ, USA). HRP substrate kit was purchased from Millipore Corporation (Billerica, MA, USA). Transwell® permeable supports were obtained from Corning Incorporated (Corning, NY, USA). DMSO was used as vehicle control for AUDA and CDU. Ethanol was used as vehicle control for EETs.
4
Lipid Mediator Synthesis and Analysis
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Arachidonic acid (5Z,8Z,11Z,14Z-eicosa-5,8,11,14-tetraenic acid) was obtained from MP Biomedicals Germany GmbH (Eschwege, Germany). The following HETEs and EETs were ordered from Cayman Chemical (Ann Arbor, Michigan, USA): (±) 18-HETE ((±) 18-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid), 19(S)-HETE (19S-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid), 19(R)-HETE (19R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid), (±) 11(12)-EET ((±) 11,(12)-epoxy-5Z,8Z,14Z-eicosatrienoic acid) and (±) 14(15)-EET ((±) 14(15)-epoxy-5Z,8Z,11Z-eicosatrienoic acid). All other chemicals were obtained from VWR International GmbH (Darmstadt, Germany) if not indicated otherwise. The chemicals used had reagent-grade purity or were analytical standards.
5
Cytotoxicity and Migration Assays
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Cell culture medium (RPMI 1640 medium) and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). The ELISA kits for 11,12-EET and 14,15-EET were obtained from Detroit R&D (Detroit, MI, USA). 11,12-EET, 14,15-EET, and 17-ODYA were purchased from Cayman Chemical (Ann Arbor, MI, USA). 3-(4, 5-dimethyl-2-thiazy1)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and DMSO were obtained from Sigma Chemical Co. (St. Louis, MO, USA). PI and Annexin V/FITC detection kits were purchased from Bender Medsystems Inc. (Burlingame, CA, USA). Antibodies against CyclinD1, Bax, Bcl-2, MMP-2, and MMP-9 were purchased from Epitomics Inc. (Burlingame, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-rabbit IgG) were purchased from KPL (Gaithersburg, MA, USA). Enhanced chemiluminescence reagents were purchased from Pierce, Inc. (Rockford, IL, USA). The Transwell plates were obtained from Corning Costar (Cambridge, MA, USA), and the Matrigel was purchased from BD Biosciences (Bedford, MA, USA).
6
Calcium Signaling in Cell Lines
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14,15-EET and 14,15-EE5ZE were from Cayman (Ann Arbor, MI). Quest Fluo-8 NW Calcium Assay Kit was from AAT Bioquest (AAT Bioquest Inc., Sunnyvale, CA, USA). Ca2+ free medium, capsaicin, capsazepine (CPZ), SB366791, HC030031 (HC), 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester (BAPTA/AM), ethylene glycol tetraacetic acid (EGTA), Griess's reagent, and Drabkin's reagent kit 525 were from Sigma-Aldrich (St. Louis, MO, USA). TurboFect was from Fermentas (Glen Burnie, MD, USA). Matrigel was from BD Biosciences (San Jose, CA, USA).
7
Splenic and Aortic Cell Analysis in Atherosclerosis
8
Eicosanoid Signaling Pathway Modulators
9
Eicosanoid Compound Procurement Protocol
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10
Hepatocyte Isolation and Insulin Stimulation
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Primary hepatocytes were isolated by collagenase/EGTA perfusion as published elsewhere (18 ) from male C3HeB/FeJ mice. Following preparation, hepatocytes were resuspended in Williams E Medium (Gibco, Paisley, UK, Catalogue Number 32551), 10% (v/v) FCS, 100 nm dexamethasone. The hepatocyte cell lines Hepa 1–6 and BNL CL.2 were obtained from ATCC (Manassas, VA) and cultured in DMEM (Gibco, Catalogue Number 41966), 10% (v/v) FCS.
For stimulation experiments, cells were seeded onto collagen I coated six-well plates and cultured in serum-free starvation medium (Williams E Medium and DMEM for primary hepatocytes and hepatocyte cell lines, respectively). Subsequently, cells were pre-treated with EETs (4 μm of 5(6)-EET, 8(9)-EET, 11(12)-EET and 14(15)-EET (Cayman Chemicals, Ann Arbor, MI) or vehicle (DMSO) in starvation medium for 1h at 37 °C 5% CO2. Next, cells were stimulated with starvation medium containing the indicated concentrations of human insulin (Sigma-Aldrich, Steinheim, Germany) with or without EETs at the same concentration as above. Stimulation media for this second step were supplemented with Phosphatase Inhibitor Mixture 2 and 3, (Sigma-Aldrich) at 1:200. DMSO was added to wells without EETs to yield the same final concentration in all wells of the experiment. Cells were incubated for 20, 40 or 60 min at 37 °C 5% CO2 and scraped into 50 μl RIPA buffer.
For stimulation experiments, cells were seeded onto collagen I coated six-well plates and cultured in serum-free starvation medium (Williams E Medium and DMEM for primary hepatocytes and hepatocyte cell lines, respectively). Subsequently, cells were pre-treated with EETs (4 μ
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