Cis 11 14 eicosadienoic acid

Lipopolysaccharide (LPS), acridine orange (AO), Triton X-100, sulfanilamide, dichlorodihydrofluorescein diacetate (DCDHF-DA), 3-(4,5-dimethylthiazolyl- 2)-2,5-diphenyltetrazolium bromide (MTT), palmitic acid (≥99%), cis 11-eicosenoic acid (≥99%) and cis 11,14-eicosadienoic acid (≥98%) were from Sigma-Aldrich. Ergosta-7,22-dien-3-ol (≥98%) was from BioBioPha Co., Ltd (China). Sodium nitroprusside dehydrate (SNP) from Riedel-de-Haën (Seelze, Germany). N-(1-naphtyl)-ethylene-diamine dihydrochloride were obtained from Merck (Darmstadt, Germany). Dulbecco’s Modified Eagle Medium (DMEM), foetal bovine serum (FBS), Dulbecco’s phosphate buffer saline (DPBS), Hank’s balanced salt solution (HBSS) and antibiotic were from GIBCO (Invitrogen, UK). COX-2, iNOS, IKB-α, β-tubulin and CHOP primary antibodies, as well as anti-rabbit secondary antibody, were from Santa Cruz, USA. IL-6 ELISA kit was from AbCam, UK.

Myristic acid (C14:0, T0502), pentadecanoic acid (C15:0, P6125), palmitic acid (C16:0, P9767), margaric acid (C17:0, H3500), stearic acid (C18:0, S4751), oleic acid (C18:1, O1008), linoleic acid (C18:2, 62240), cis-11-eicosenoic acid (C20:1, E3635), and cis-11,14-eicosadienoic acid (C20:2, E3127) were purchased from Sigma-Aldrich. These LCFAs were conjugated with non-esterified fatty acid (NEFA)-free BSA (Sigma-Aldrich, a7030). Briefly, fatty acids were dissolved in sterile water using a vortex and heated to 70 °C for 10 min. Fatty acids were conjugated to BSA in serum-free RPMI containing 5% NEFA-free BSA immediately after dissolving as described previously^{41 (link)}. The conjugated-fatty acids were shaken at 140 rpm at 40 °C for 1 h before adding to the cells. Serum-free RPMI containing 5% NEFA-free BSA was used as the vehicle control. To evaluate the effects of palmitic acid in vivo, mice were injected with palmitic acid (15 μM) conjugated to free NEFA-free BSA. The fatty acid concentration in mouse serum after palmitic acid injection was measured using the acyl-CoA synthetase-acyl-CoA oxidase method (HR series NEFA HR, Wako).

The following fatty acid and fatty acid methyl ester standards were purchased from Sigma Aldrich (Gillingham, Dorset, UK): tricosanoic acid (C23:0), behenic acid (C22:0), cis-11,14-eicosadienoic acid (C20:2 Δ11,14), heptadecanoic acid (C17:0), cis-10-pentadecanoic acid (C15:1Δ10), pentadecanoic acid methyl ester (C15:0) (PDA), palmitic acid methyl ester (C16:0), palmitoleic acid methyl ester (C16:1Δ9), cis-10-heptadecanoic acid methyl ester (C17:1Δ10), stearic acid methyl ester (C18:0), elaidic acid methyl ester (t-C18:1Δ9), oleic acid methyl ester (C18:1Δ9), linolelaidic acid methyl ester (t-C18:2 Δ9, 12) linoleic acid methyl ester (C18:2 Δ9,12), arachidic acid methyl ester (C20:0), linolenic acid methyl ester (C18:3 Δ9,12,15), heneicosanoic acid methyl ester (C21:0), all-cis-11,14,17-eicosatrienoic acid methyl ester (C20:3 Δ11,14,17), arachidonic acid methyl ester (C20:4 Δ5,8,11,14), all-cis-13,16-docosadienoic acid methyl ester (C22:2 Δ13,16), lignoceric acid methyl ester (C24:0), nervonic acid methyl ester (C21:1 Δ15), docosahexanoic acid methyl ester (C22:6 Δ4,7,10,13,16,19) (DHA), all-cis-8,11,14-eicosaterinoic acid methyl ester (C20:3 Δ8,11,14), erucic acid methyl ester (C22:1 Δ13). All solvents used (methanol n-hexane and dichloromethane) were CHROMASOLV HPLC grade (Sigma Aldrich, UK). Hydrogen chloride (1.25 M) in methanol was purchased from Fluka Analytical (UK).