Lipids and lipid standards were purchased from Avanti Polar Lipids (Alabaster, Alabama) or Cayman Chemical (Ann Arbor, Michigan). Deuterated standards are as follows: PGE2-d4: 9-oxo-11α,15S-dihydroxy-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acid, ≥99% deuterated forms. HPLC grade solvents were from Thermo Fisher Scientific (Hemel Hempstead, Hertfordshire UK). PAR-1 and −4 agonists and U73112, U73343 were from Tocris Biosciences (Bristol, UK). COX-1 inhibitor (Sc-560) was from Cayman Chemical. Platelet signaling inhibitors (oleyloxyethylphosphocholine (OOEPC), bromoenol lactone (BEL), cytosolic phospholipase A2α (cPLA2α) inhibitor (N-((2S,4R)−4-(Biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenyl]acrylamide, HCl), Gö 6850 and wortmannin were from Calbiochem (UK). All other reagents were from Sigma-Aldrich unless otherwise stated. Ovine COX-1 was from Cayman Chemical or purified as described [9] (link), [10] (link).
Ovine cox 1
Manufactured by Cayman Chemical
Ovine COX-1 is a laboratory equipment product manufactured by Cayman Chemical. It is an enzyme that plays a key role in the synthesis of prostaglandins and other eicosanoids. The Ovine COX-1 product is derived from ovine (sheep) sources and is commonly used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Human recombinant cox 2, by Cayman Chemical (3 mentions)
Cox1 inhibitor sc 560, by Cayman Chemical (1 mentions)
Hplc grade solvents, by Thermo Fisher Scientific (1 mentions)
Sep pak c18 35 cc vac cartridges, by Waters Corporation (1 mentions)
Nova pak c18 radial pak column, by Waters Corporation (1 mentions)
4 protocols using ovine cox 1
1
Lipid and Signaling Pathway Inhibitors
2
Determination of COX-1 and COX-2 Activity
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For the determination of cell-free COX activity, isolated ovine COX-1 (50 units, Cayman Chemicals) or human recombinant COX-2 (20 units, Cayman Chemicals) were pre-treated with vehicle (DMSO) or test compounds in 100 mmol/L Tris pH 8 (supplemented with 5 mmol/L glutathione, 5 μmol/L hemoglobin and 100 μmol/L EDTA) for 5 min at 4 °C. The mixture was pre-warmed for 1 min at 37 °C, and arachidonic acid was added (5 μmol/L for COX-1 and 2 μmol/L for COX-2). The reaction was stopped by addition of an equal volume of ice-cold methanol, and COX-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid (12-HHT) was extracted by SPE using Sep-Pak C18 35 cc Vac Cartridges (Waters) and analyzed by RP-UV‒HPLC on a Nova-Pak C18 Radial-Pak Column as described for 5-LOX products. The COX-1/2 inhibitor indomethacin (10 μmol/L) was used as control.
For the determination of cellular COX-1 activity, freshly isolated human platelets (1 × 108) were pre-treated with vehicle (DMSO), test compounds or indomethacin (10 μmol/L) for 5 min at room temperature, and arachidonic acid (5 μmol/L) was added. After 5 min at 37 °C, the reaction was stopped by addition of an equal volume of ice-cold methanol, and formed 12-HHT was extracted and analyzed as described above.
For the determination of cellular COX-1 activity, freshly isolated human platelets (1 × 108) were pre-treated with vehicle (DMSO), test compounds or indomethacin (10 μmol/L) for 5 min at room temperature, and arachidonic acid (5 μmol/L) was added. After 5 min at 37 °C, the reaction was stopped by addition of an equal volume of ice-cold methanol, and formed 12-HHT was extracted and analyzed as described above.
3
Inhibition of Ovine COX-1 and Human COX-2
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Inhibition of ovine COX-1 and human COX-2 was investigated as previously described (Siemoneit et al., 2008 (link)). Briefly, purified ovine COX-1 (50 units, Cayman Chemical, Ann Arbor, MI) or human recombinant COX-2 (20 units, Cayman Chemical, Ann Arbor, MI) were diluted in 1 ml of reaction mixture containing 100 mM Tris buffer pH 8, 5 mM glutathione, 5 µM hemoglobin, and 100 µM EDTA at 4°C and pre-incubated with the test compound for 5 min. Samples were pre-warmed for 60 s at 37°C, and AA (5 µM for COX-1 and 2 µM for COX-2) was added to start the reaction. After 5 min at 37°C, the reaction was stopped, PGB1 as standard was added and 12-HHT was extracted and then analyzed by HPLC (Siemoneit et al., 2008 (link)). Indomethacin (10 µM) was used as a well-recognized reference inhibitor of COX-1 and celecoxib for COX-2 to control the assays.
4
Determination of COX-1 and COX-2 Activities
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For determination of COX activities, purified ovine COX-1 (Cayman Chemicals; 50 units) or human recombinant COX-2 (Cayman Chemicals; 20 units) were diluted in Tris buffer (100 mM, pH 8) supplemented with glutathione (5 mM), EDTA (100 µM) and hemoglobin (5 µM). After pre-incubation with TWG, vehicle (0.1% DMSO) or indomethacin (IND, 10 µM) for 5 min at RT, the samples were pre-warmed for 30 s at 37 °C, and the reactions were started by addition of 5 µM AA (COX-1) or 2 µM AA (COX-2). After 5 min at 37 °C the reactions were stopped by addition of one volume of ice-cold methanol. Formation of COX-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid (12-HHT) was analyzed by RP-HPLC on a Nova-Pak C18 Radial-Pak Column (4 µm, 5 × 100 mm, Waters) as described [31 (link)].
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