Hils (2.8 ?106 cells/mL) were incubated (37 ) with RCM-101 extract, NDGA (as a
Hils (2.8 ?106 cells/mL) were incubated (37 ) with RCM-101 extract, NDGA (as a positive control, 0.1, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27741243 1 or 10 g/mL) or vehicle (ethanol), for 5 minutes before the addition of arachidonic acid (2.5 M) substrate. Porcine neutrophils were suspended in Hanks’ buffer in concentration of 2.8 ?106 cells/mL. RCM-101 (0.1, 1, 100 g/ mL) was added 10 minutes before the calcium ionophore A23187 (2.5 M). After 5 minute incubation, production of LTB4 was initiated by the addition of the calcium ionophore A23187 (2.5 M) and 5 minutes later the reaction was terminated by adjusting the pH to 3 with citric acid. PGB2 (45 ng) and 15-HETE (83 ng) were then added as internal standards. The reaction mixture was extracted with 5 mL of chloroform/methanol (7:3 v/v) and dried under vacuum. The residue was dissolved in 120 L of HPLC mobile phase (methanol-water-acetic acid, 76/34/ 0.08, v/v/v, pH 3.0) and leukotriene metabolites were assayed using a Waters HPLC system equipped with an auto sampler, a multi-solvent delivery system and a Waters 996 Photodiode Array Detector. Standard curves were prepared by the addition of LTB4 (10 ?200 ng) and 5-HETE (500 ?800 ng) to neutrophil suspensions. Data were analysed using Water Millenium Software, Version 3.2, results being expressed as percentage of the vehicle control which was taken as 100 .Prostaglandin E2 production Murine macrophages (Raw 264.7 cells, American Type Culture Collection, Rockville, MD, USA) were grown in RPMI 1640 medium, supplemented with 10 heat-inactivated FBS, 100 g/mL gentamycin, 1.5 g/L sodium bicarbonate and 10 mM HEPES, at 37 , in an atmosphere containing 5 CO2. Cells were sub-cultured once a week by harvesting them with trypsin/EDTA and seeding them in 75 cm2 flasks. Once confluent, murine macrophages were suspended in serum-free RPMI medium at concentration of 2 ?105 cells/mL, and the cells were seeded in 24-well plates (1 ?105 cells/well), in serum-free RPMI medium. Cells were then treated with RCM-101 (1, 10, or 100 g/mL) or vehicle 10 minutes before the addition of LPS (1 g/mL). The supernatant and the cells were separated. PGE2 was assayed in the supernatant using an immune-enzyme analysis kit. The assay depends on competition between PGE2 and PGE2acetylcholinesterase conjugate (PGE2-tracer) for a limited amount of monoclonal PGE2antibody. The assays were carried out according to the manufacturer’s protocol, in triplicate. PGE2 release was calculated using software supplied by the kit manufacturer. Determination of COX-1 and COX-2 protein expression in Raw 264.7 cells Cultured Raw 264.7 cells prepared as described above for determination of PGE2 production, with and without incubation with RCM-101, were washed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 twice with icecold phosphate buffer saline then lysed with 100 L/well of lysis buffer (50 mM Tris base, pH 7.6, 2 mM MgCl2, 1 mM EGTA, 1 TritonX, 1 mM phenyl PMSF, 1 mM pepstatin, 1 mM aprotinin, 1 mM leupeptin) for 5 minutes. The cells and the supernatant were collected and centrifuged for 5 minutes at 14000 rpm. The cell debris was 4F-Benzoyl-TN14003 custom synthesis discarded and the supernatant was assayed for protein concentration using Coomassie Protein Assay Kit (BioRad Laboratories Pty Ltd, California, USA) and the UV-visible spectrophotometer (Cintra 5, GBC Scientific Equipment Pty Ltd, Illinois, USA)COX-1 and COX-2 protein was measured by Western blotting as previously described [16] with a slight modification. Aliquots of 20 g of total protein were loaded to each lane of 7.5 SDS-polyacrylamide gels.
