After cooling in an icebox, 0.5 mL of 14% methanol BF3 (Aldrich Chemistry, St. Louis, MO, USA) was added and the mixture was heated at 80°C in the heating block for 30 min. After cooling, 1 mL of n-hexane high performance liquid chromatography (HPLC) grade] and 0.5 mL of H2O (HPLC grade) were added to the reaction mixture. The supernatants were collected and a small amount PLX3397 mouse of Na2SO4 was added to remove the H2O. The solutions were filtered using a syringe filter (0.2 μm, 13 mm) and stored at −4°C until GC/MS analysis. A DB-5 column (0.25-μm film thickness × 0.25 mm diameter × 30 m length) was used for the GC/MS experiment. Helium was used as the carrier gas at a flow rate

of 23.3 mL/min. The oven temperature was programmed as follows: 80°C for 2 min, increased to 320°C at a rate of 15°C/min and held for 10 min. The injector and detector temperatures were set at 280°C and 250°C, respectively. Sample solutions (1 μL) were injected into the GC column with a 10.0 split ratio. Detection was performed by electron ionization (70 eV) and quadrupole mass spectrometry. E7080 molecular weight Fatty acids were identified by comparing their mass spectra with those of a library (Wiley Library, version

2008; John Wiley & Sons Inc., Hoboken, NJ, USA). Murine macrophage RAW264.7 cells (Korea Cell Line Bank, Seoul, Korea) were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 2mM glutamate, 100 units/mL of penicillin, and 100 μg/mL of streptomycin (WelGENE Inc., Seoul, Korea) in a humidified incubator with 5% CO2. The amount of NO was calculated by measuring the amount of nitrite, an oxidized product, in the cell culture supernatants as previously explained [18]. RAW264.7 cells were seeded in 96-well cell culture plates at a density of 1 × 104 cells/well and incubated for 12–18 h. After discarding the growth medium, cells were stimulated with 1 μg/mL LPS (Sigma-Aldrich Co., St. Louis, MO, USA) in the presence

of various ADP ribosylation factor concentrations of each compound in a serum-free medium for 20 h. Next, 100 μL of cell culture supernatant was mixed with 100 μL of Griess reagent (Sigma-Aldrich Co.) in a new 96-well plate, followed by spectrophotometric measurement at 550 nm according to the manufacturer’s instructions (BioTek Instruments, Inc., Winooski, VT, USA). Nitrite concentrations were determined by comparison with a sodium nitrite standard curve. RAW264.7 cells were seeded in 96-well cell culture plates at a density of 1 × 104 cells/well and incubated for 12–18 h. After discarding the growth medium, cells were treated with various concentrations of each compound in a serum-free medium for 20 h. After treatment, 10 μL of 10 μg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich Co.) solution was added to each well (except for the blank well), and the sample was reincubated in an incubator (at 37°C, 5% CO2) in darkness.