Pge2 kit

A PGE₂ determination is an assay which detects the reaction between PGE₂ and PGE₂-acetylcholinesterase (AchE) conjugate, identified as the PGE₂ tracer. The supernatant obtained following the homogenization and centrifugation of gastric ulcer tissue samples were subjected to PGE₂ activity determination using the PGE₂ kit from Cayman Chemical Company (USA). Using the ELISA reader (Asys UVM 340, UK), the absorbance that represents the PGE₂ activity was determined at the wavelength of 405 and 420 nm. The results were expressed as pg/mL protein.

PC (Parry India Ltd, India) was dissolved in phenol red free DMEM to prepare a stock solution (200 μM) and stored at 4 °C. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and 0.25 % Trypsin-EDTA were procured from Invitrogen (Carlsbad, CA). Supplements for cell culture were purchased from Sigma Aldrich (St. Louis, MO) and Hi-media. (Mumbai, India) Neutral red was purchased from Sigma Aldrich (St. Louis, MO). Annexin V-FITC Apoptosis Detection Kit was purchased from BD Biosciences (San Jose, CA). The inhibitor cocktail was obtained from Pierce (Rockford, IL) and antibodies against ERK1/2, γH2AX (Ser 139), COX-2, cytochrome C, p-65, AKT and Vinculin were from Cell Signaling Technology (Beverly, MA). Respective secondary mouse, rabbit and goat antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence solution was from GE health care. Primers for Cyclin E, CDK-2, p-21, Bax, Bcl-2, Caspase 9, Mcl-1,MMP-9,VEGFR-2 and GAPDH were obtained from Eurofins (Luxembourg, Europe). C-DNA reverse transcriptase kit was procured from ABI (Carlsbad, CA) and SYBR green PCR master mix was from Roche (BASEL, Switzerland). Alexa Flour was from Life Technologies (Carlsbad, CA). PGE-2 kit was obtained from Cayman chemicals (Ann Arbor, MI). Matrigel was obtained from BD Biosciences (San Jose, CA, USA).

IL-1ß, IL-6, IL-8 and PGE₂ were measured in the FLS culture media using commercially available ELISA kits (IL-1ß Quantikine® ELISA from R&D Systems, Wiesbaden, Germany, IL-6 High Sensitivity Kit from eBioscience, San Diego, USA; Human CXCL8/IL-8 Immunoassay from R&D Systems, Wiesbaden, Germany; PGE₂ Kit from Cayman Chemical Company, Ann Arbor, USA) according to the manufacturer’s instructions. TNF-α was quantified using two ELISA kits with different sensitivities (Human TNF-α kit from R&D Systems, Wiesbaden, Germany and Human TNF-α UltraSensitive Kit from Invitrogen™, Karlsruhe, Germany). Cultured medium to be analysed was concentrated fivefold using a centrifugal filter (Amicon^® Ultra-0.5 ml 3K-device from Merck, Darmstadt, Germany) to allow small amounts of TNF-α to be detected. All data were normalised with respect to the cellular protein content.