Cox inhibitor screening kit

All chemicals, reagents used in this project were purchased from Sigma-Aldrich Co., (St Louis, MO, USA), Merck Millipore (Billerica, MA, USA) and Acros Organics (Thermo Fisher Scientific, Waltham, MA, USA) (above 98% purity) and used without additional purification. The chemicals used in sPLA₂ assay comprised of 1,2-bis(heptanoylthio)-phosphatidylcholine, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), and recombinant human phospholipase A₂ (PLA₂)-V from Cayman Chemicals (Ann Arbor, MI, USA). CaCl₂, KCl, and HCl were procured from Merck Millipore. COX inhibition activity was determined using a COX Inhibitor Screening Kit purchased from Cayman Chemicals. The levels of TNF-α and IL-6 in the media were calculated by an enzyme-linked immunosorbent assay (ELISA) using mouse TNF-α (catalogue no 500850) and mouse IL-6 (catalogue no 583371), ELISA kits were obtained from Cayman Chemicals. Mouse RAW264.7 macrophages were obtained from Abcam plc (Cambridge, UK). Cell culture chemicals and fetal bovine serum (FBS) were obtained from Sigma-Aldrich Co., and FBS was heat-inactivated for 30 minutes at 65°C. LPS supplied by Sigma-Aldrich Co., was dissolved in phosphate buffered saline (PBS). The synthetic compounds were dissolved in dimethyl sulfoxide (DMSO).