Thromboxane b2 elisa kit

Urethane, norepinephrine bitartrate, phenylephrine, sodium nitroprusside, acetylcholine, dimethyl sulfoxide (DMSO), indomethacin, ethylenediaminetetraacetic acid (EDTA), terutroban, acetylsalicylic acid, 1-benzylimidazole, and kaempferol were purchased from Sigma-Aldrich (Prague, Czech Republic). The Krebs solution salts and 96% ethanol were purchased from Penta s.r.o. (Prague, Czech Republic). Arachidonic acid was bought from Medista (Prague, Czech Republic). Heparin sodium was purchased from Zentiva (Prague, Czech Republic); U-46619, thromboxane B₂ ELISA kit, prostaglandin H₂, and the COX inhibitor screening kit were from the Cayman Chemical Company (Ann Arbor, MI, USA). Collagen was obtained from Diagnostica a.s. (Prague, Czech Republic), and saline from B. Braun (Prague, Czech Republic).

We assessed eicosanoid production by chondrocytes using the Prostaglandin E₂ ELISA Kit – Monoclonal, Leukotriene B₄ ELISA Kit and Thromboxane B₂ ELISA Kit, according to the manufacturer's recommendations (Cayman Chemical, Ann Arbor, MI, USA). The concentration of each eicosanoid was determined according to the manufacturer's recommendations.

Cells were seeded in 6-well culture plates at 2 × 10⁵ cells/well. After incubating for 24 hours, cells were treated with two different concentrations of celecoxib or mPGES-1 inhibitor CAY10678 (Cayman Chemical) or 0.1% DMSO and incubated for another 24 hours. Cell culture supernatants were collected and centrifuged at 3,000 × g for 10 minutes. Tumor lysates (100 mg) were washed three times with PBS and prepared in 500 μL RIPA buffer supplemented with 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail (Thermo Fisher Scientific). Then, the tissue samples were sonicated with a sonicator thoroughly on ice, followed by centrifugation at 10,000 × g for 20 minutes. Protein concentration was determined, and the tumor tissue lysates were analyzed for ELISA. Levels of released prostaglandins and thromboxane in supernatants and tumor tissues were quantified using Prostaglandin E2 ELISA kit, Prostaglandin D2 ELISA kit, Prostaglandin F2α ELISA kit, 6-keto Prostaglandin F1α ELISA kit, and Thromboxane B2 ELISA kit (Cayman Chemical), according to the manufacturer's recommendations.