Pge2 d4

Manufactured by Cayman Chemical

Sourced in United States, Germany

PGE2-d4 is a stable-isotope labeled internal standard for the quantification of prostaglandin E2 (PGE2) by mass spectrometry. It serves as a reference compound to ensure accurate and reliable measurement of PGE2 levels in biological samples.

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Lab products found in correlation

52 protocols using pge2 d4

Eicosanoid Profiling in Cell Culture

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HPLC-grade water, methanol, acetonitrile, and isopropyl alcohol were purchased from J.T. Baker (Avantor Performance Material, Inc., Center Valley, PA, USA). Acetic acid and ammonium acetate were obtained from Sigma-Aldrich (St. Louis, MO, USA). The eicosanoid standards were as follows: 14,15-EET-d11, 5(S)HETE-d8, LTB₄-d4, PGE₂-d4, PGD₂-d4, and AA-d8 (Cayman Chemical, Ann Arbor, MI, USA). A Strata-x 33-µm polymerized solid reverse-phase extraction column (cat # 8B-S100-UBJ) was purchased from Phenomenex (Torrance, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-ethylenediaminetetraAcetic acid (EDTA) were purchased from HyClone Laboratories Inc. (Logan, UT, USA). Escherichia coli LPS and Griess reagent were obtained from Sigma Chemical Co. (St Louis, MO, USA).

Lee J.W., Mok H.J., Lee D.Y., Park S.C., Ban M.S., Choi J., Park C.G., Ahn Y.S., Kim K.P, & Kim H.D. (2016). UPLC-MS/MS-Based Profiling of Eicosanoids in RAW264.7 Cells Treated with Lipopolysaccharide. International Journal of Molecular Sciences, 17(4), 508.

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Quantitative Eicosanoid Profiling by UPLC-MS/MS

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Cell-free culture media (0.5 ml) were mixed with 2 ng of deuterated internal standard solutions, centrifuged (12000× g, 3 min), mixed with 6 ml of 0.1% acetic acid, and loaded onto Oasis® PRiME hydrophilic lipophilic balance (HLB) solid-phase lipid extraction cartridge (60 mg, 3cc, (Waters, Milford, MA, USA) catalogue number 186008056). The cartridge was washed with 2 ml of 15% methanol containing 0.1% formic acid and the lipids were sequentially eluted with 500 μl of anhydrous methanol and 500 μl of acetonitrile. The resulting samples were mixed, concentrated by evaporation of the solvent under gentle stream of nitrogen, and stored at −80 °C. The target eicosanoids were identified by triple quadrupole UPLC-MS/MS (Shimadzu 8040, Kyoto, Japan) under previously reported run conditions [17 (link)] and quantified by comparing their MS, MS/MS, and UPLC (retention times, peak areas) data with the data obtained for deuterated internal standard compounds (6-keto PGF_1α-d4 (cat.no. 315210), TXB₂-d4 (cat.no. 319030), PGF_2α-d4 (cat.no. 316010), PGE₂-d4 (cat.no. 314010), PGD₂-d4 (cat.no. 312010), LTB₄-d4 (cat.no. 320110), 12(S)-HETE-d8 (cat.no. 334570) (Cayman Chemical, Ann Arbor, MI, USA) of the same classes using Lipid Mediator Version 2 software package (Shimadzu, Kyoto, Japan).

Astakhova A., Chistyakov D., Thomas D., Geisslinger G., Brüne B., Sergeeva M, & Namgaladze D. (2019). Inhibitors of Oxidative Phosphorylation Modulate Astrocyte Inflammatory Responses through AMPK-Dependent Ptgs2 mRNA Stabilization. Cells, 8(10), 1185.

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Prostaglandin Synthase Activity Assay

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Prostaglandin-endoperoxide synthase 1 (Ovine, ≥90 % purity) and 2 (Human Recombinant, ≥70 % purity) were purchased from Cayman (Ann Arbor, MI, USA) and used within 2 weeks of delivery. Enzyme activity was confirmed by an oxygen consumption assay. Arachidonic acid, AAPH, PGH₂, 8-iso-PGF_2α, PGF_2α, PGE₂, PGA₂, 8-iso-PGF_2α-d₄, PGF_2α-d₄, and PGE₂-d₄ were also purchased from Cayman. Tris-HCl, phenol, porcine hematin, indomethacin, meclofenamic acid, trolox [(±)-6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxylic acid], tin (II) chloride dihydrate, diethyl ether, ethanol, and acetic acid were purchased from Sigma Aldrich (St. Louis, MO, USA).

van ‘t Erve T.J., Lih F.B., Kadiiska M.B., Deterding L.J., Eling T.E, & Mason R.P. (2015). Reinterpreting the Best Biomarker of Oxidative Stress: The 8-iso-PGF2α / PGF2α Ratio Distinguishes Chemical from Enzymatic Lipid Peroxidation. Free radical biology & medicine, 83, 245-251.

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Quantification of Lipid Mediators

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Prostaglandins: PGE₂ (99%), PGE₃ (98%); prostacyclins: 6-keto-PGF_1α (98%), Δ17-6-keto-PGF_1α (98%); leukotriene: LTB₄, (97%); resolvins: RvD₁ (95%), RvD₂ (95%), RvD₃ (95%), RvD₄ (95%), RvD₅ (95%) and deuterated internal standards: PGE₂-d₄ (99%), 6-keto-PGF_1α-d₄ (99%), LTB₄-d₄ (97%) and RvD₂-d₅ (95%) were purchased from Cayman Chemical (Ann Arbor, MI, USA).
Acetonitrile (99.8%), ethanol (99.8%) acetic acid (99%) and formic acid (98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA), 2-propanol (HPLC grade, 99.9%) from Merck (Darmstadt, Germany), a Millipore Milli-Q system was used to produce ultra-pure water 18 MΩ (Millipore, Milford, MA, USA). EPA (99%), ARA (95%), DHA (98%) and ALA (99%) were from Sigma-Aldrich (Oslo, Norway). Complete EBM-2 contained EBM^TM-2 basal medium supplemented with 0.1% heparin, 0.1% R³-IGF−1 solution, 0.1% ascorbic acid, 0.04% hydrocortisone, 0.4% h-FGF-B, 0.1% h-EGF, 0.1% GA−1000 and 2% fetal bovine serum (FBS, cat# 14-801F) was from BioWhittaker (Petit Rechain, Belgium).

Araujo P., Belghit I., Aarsæther N., Espe M., Lucena E, & Holen E. (2019). The Effect of Omega-3 and Omega-6 Polyunsaturated Fatty Acids on the Production of Cyclooxygenase and Lipoxygenase Metabolites by Human Umbilical Vein Endothelial Cells. Nutrients, 11(5), 966.

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Serum Lipid and Eicosanoid Analysis

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Blood was collected during terminal procedures after fasting (5 h) and spun to isolate serum, then stored at −80 °C. Serum samples were subsequently analyzed for insulin and IL-10 levels using ELISAs (Millipore and R&D Systems, respectively) or total cholesterol and triglycerides (Sigma). For eicosanoid analysis, PGE₂ was extracted from serum in methanol and isolated by C18 solid-phase extraction chromatography. PGE₂-d₄ (Cayman Chemicals, Ann Arbor, MI, USA) was added to the experimental system as an internal standard. PGE₂ was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a TSQ Quantum Ultra triple quadrupole mass spectrometer coupled to an Accela 1250 UHPLC system (Thermo Scientific, Hemel Hempstead, UK) in negative ion mode with a C18 column and methanol-water gradient. Quantification was performed using the multiple reaction monitoring (MRM) mode using the transitions PGE₂ (351 → 271) and PGE₂-d₄ (355 → 193). The concentration of PGE₂ was determined by comparison to a calibration curve run in parallel.

Thompson D., Morrice N., Grant L., Le Sommer S., Ziegler K., Whitfield P., Mody N., Wilson H.M, & Delibegović M. (2017). Myeloid protein tyrosine phosphatase 1B (PTP1B) deficiency protects against atherosclerotic plaque formation in the ApoE−/− mouse model of atherosclerosis with alterations in IL10/AMPKα pathway. Molecular Metabolism, 6(8), 845-853.

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Quantitative Analysis of Prostaglandin E2

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Stool ethanol extracts (30 μl) were mixed with 90 μl methanol containing PGE2-d4 as an internal standard (Cayman Chemical Co.) and centrifuged (10 min, 9000 g, 4 °C). The supernatants (10 μl) were injected onto a 150 × 2.1-mm ACQUITY BEH C18 column (Waters). The column was eluted isocratically at a flow rate of 450 μl/min with 20% mobile phase A (0.01% formic acid in water) for 3 min followed by a linear gradient to 100% mobile phase B (0.01% acetic acid in acetonitrile) over 12 min. MS analyses were carried out using electrospray ionization in the negative ion mode using full scan analysis over m/z 70–850 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings are: ion spray voltage, –3.5 kV; capillary temperature, 320 °C; probe heater temperature, 300 °C; sheath gas, 45; auxiliary gas, 10; and S-lens RF level 60.

Ma W., Wang Y., Nguyen L.H., Mehta R.S., Ha J., Bhosle A., Mclver L.J., Song M., Clish C.B., Strate L.L., Huttenhower C, & Chan A.T. (2024). Gut microbiome composition and metabolic activity in women with diverticulitis. Nature Communications, 15, 3612.

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Prostanoid and Nucleotide Quantification

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All prostanoids (PGE₃, PGF_3α, TXB₃, PGE₂, PGF_2α, and TXB₂), deuterated prostanoids (PGE₂-d4, PGF_2α-d4, and TXB₂-d4), and EPA were purchased from Cayman Chemical Co. (Ann Arbor, MI). Calcium ionophore A23187 and indomethacin were purchased from Sigma Aldrich (St. Louis, MO). Adenosine 5′-triphosphate (ATP) disodium salt hydrate, 5′-adenylic acid (AMP), and sodium creatine phosphate hydrate were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Creatine kinase was purchased from Roche Applied Science (Tokyo, Japan). Dipyridamole, probenecid, quercetin, methotrexate, and folic acid were purchased from Wako Pure Chemical Industries (Osaka, Japan). MK571, celecoxib, candesartan, estradiol 17β-glucronide (E₂17βG), adenosine 3′, 5′-cyclic monophosphate (cAMP), and guanosine 3′, 5′-cyclic monophosphate (cGMP) were purchased from Sigma Aldrich (St. Louis, MO). All other chemicals were of the highest purity available.

Tanaka N., Yamaguchi H, & Mano N. (2014). Transport of Eicosapentaenoic Acid-Derived PGE3, PGF3α, and TXB3 by ABCC4. PLoS ONE, 9(10), e109270.

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Quantitative Profiling of Prostaglandin E2 in Stool Samples

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Stool homogenates (30 μL) were extracted using 90 μL of methanol containing PGE2d4 as an internal standard (Cayman Chemical Co.; Ann Arbor, MI) and centrifuged (10 min, 9,000 x g, 4°C). The supernatants (10 μL) were injected onto a 150 × 2 mm ACQUITY T3 column (Waters; Milford, MA). The column was eluted isocratically at a flow rate of 400 μL/min with 25% mobile phase A (0.1% formic acid in water) for 1 minute followed by a linear gradient to 100% mobile phase B (acetonitrile with 0.1% formic acid) over 11 minutes. MS analyses were carried out using electrospray ionization in the negative ion mode using full scan analysis over m/z 200550 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings were: ion spray voltage, 3.5 kV; capillary temperature, 320°C; probe heater temperature, 300 °C; sheath gas, 45; auxiliary gas, 10; and Slens RF level 60.

Franzosa E.A., Sirota-Madi A., Avila-Pacheco J., Fornelos N., Haiser H.J., Reinker S., Vatanen T., Brantley Hall A., Mallick H., McIver L.J., Sauk J.S., Wilson R.G., Stevens B.W., Scott J.M., Pierce K., Deik A.A., Bullock K., Imhann F., Porter J., Zhernakova A., Fu J., Weersma R.K., Wijmenga C., Clish C.B., Vlamakis H., Huttenhower C, & Xavier R.J. (2018). Gut microbiome structure and metabolic activity in inflammatory bowel disease. Nature microbiology, 4(2), 293-305.

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Quantification of Prostaglandin E2 in Stool

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Stool homogenates (30 μL) were extracted using 90 μL of methanol containing PGE2-d4 as an internal standard (Cayman Chemical Co.; Ann Arbor, MI) and centrifuged (10 min, 9,000 x g, 4°C). The supernatants (10 μL) were injected onto a 150 x 2 mm ACQUITY T3 column (Waters; Milford, MA). The column was eluted isocratically at a flow rate of 400 μL/min with 25% mobile phase A (0.1% formic acid in water) for 1 minute followed by a linear gradient to 100% mobile phase B (acetonitrile with 0.1% formic acid) over 11 minutes. MS analyses were carried out using electrospray ionization in the negative ion mode using full scan analysis over m/z 200–550 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings were: ion spray voltage, −3.5 kV; capillary temperature, 320°C; probe heater temperature, 300°C; sheath gas, 45; auxiliary gas, 10; and S-lens RF level 60.

Tanes C., Bittinger K., Gao Y., Friedman E.S., Nessel L., Paladhi U.R., Chau L., Panfen E., Fischbach M.A., Braun J., Xavier R.J., Clish C.B., Li H., Bushman F.D., Lewis J.D, & Wu G.D. (2021). Role of Dietary Fiber in the Recovery of the Human Gut Microbiome and its Metabolome. Cell host & microbe, 29(3), 394-407.e5.

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Quantification of Lipid Mediators

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Authentic reference standards for PGE2, PGD2, PGF2α, LTB4, TXB2 and 2-AG and their deuterated analogs (PGE2-d4, PGD2-d4, LTB4-d4 and TXB2-d4) were obtained from Cayman Chemical (MI, USA) except for 2-AG whose deuterated analog (2-AG-d8) was obtained from Abcam (MA, USA). Hepes balanced salt solution and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco, Life Technologies (NY, USA). Fetal bovine serum (FBS) and all other cell culture reagents were obtained from Atlanta Biologicals (GA, USA). LC–MS/MS grade acetonitrile and methanol were obtained from Fisher Scientific (NJ, USA). Formic acid was obtained from Sigma-Aldrich (MO, USA). Deionized water was produced in-house using a Milli-Q Gradient water purification system (MA, USA).

Gandhi A.S., Budac D., Khayrullina T., Staal R, & Chandrasena G. (2017). Quantitative analysis of lipids: a higher-throughput LC–MS/MS-based method and its comparison to ELISA. Future Science OA, 3(1), FSO157.

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