Pge2 enzyme linked immunosorbent assay elisa kit

HTSMCs were grown in 6-well culture plates. When the cells reached confluence, they were exposed to thrombin for specific time periods at 37 °C. Afterward, the culture media were collected and stored at −80 °C until the assay was conducted. The levels of PGE₂ were determined using a PGE₂ enzyme-linked immunosorbent assay (ELISA) kit from Enzo Life Sciences (Lausen, Switzerland), following the manufacturer’s instructions.

The anti-inflammatory activities of the compounds were evaluated in RAW 264.7 cells cultured in DMEM with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin, and maintained at 37 °C in a 5% CO₂ atmosphere. RAW 264.7 cells (4.0 × 10⁵ cells/mL) were seeded in 96-well plates and co-treated with LPS (500 ng/mL) and the test samples. After 24 h, NO production was determined with Griess reagent, and the absorption was detected at 530 nm. The inhibitory percentage of NO production was presented as an anti-inflammatory activity. Prostaglandin E₂ (PGE₂) concentrations in the cell culture media were determined using a PGE₂ enzyme-linked immunosorbent assay (ELISA) kit (Enzo Life Sciences, Farmingdale, NY, USA). The survival rate of RAW 264.7 cells was detected by an MTT assay.

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.