Nitrite standard

Vanillylamine, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), triethylamine and 2-methyl-2-butanol were purchased from Sigma-Aldrich (Schnelldorf, Germany). Novozym^®435 was from Novozymes (Bagsværd, Denmark). n-Hexane, acetone and methanol (analytical grade) were purchased from Carlo Erba Reagenti (Milan, Italy). Roswell Park Memorial Institute (RPMI)-1640, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), l-glutamine, sodium pyruvate, β-mercaptoethanol, and glucose were acquired from Thermofisher Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, streptomycin and penicillin were purchased from Lonza (Verviers, Belgium). LPS (E. coli O111:B4) was purchased from Sigma-Aldrich (Schnelldorf, Germany). Capsaicin, Griess reagents and nitrite standard were obtained from Cayman Chemical (Ann Arbor, MI, USA). The ELISA kit for determination of insulin was from Calbiotech Inc. (Spring Valley, CA, USA). The Ca²⁺ quantification kit was from Diagnosticum Rt (Budapest, Hungary). ATP was assayed with a bioluminescence kit from Thermofisher Scientific (Waltham, MA, USA). The ELISA kits for determination of MCP-1, and CCL20 were purchased from R&D Systems (Abingdon, UK).

Eicosapentaenoic acid, Griess reagents, and nitrite standard were obtained from Cayman Chemical (Ann Arbor, MI, USA). Novozym®435 (consisting of immobilized Candida antarctica Lipase B) was supplied by Novozymes A/S (Bagsværd, Denmark). Lipopolysaccharide (O111:B4; LPS), dopamine hydrochloride, 2-methyl-2-butanol, triethylamine, phorbol 12-myristate 13-acetate (PMA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), dimethyl sulphoxide (DMSO), glutamine, HEPES and 2-mercaptoethanol were from Sigma-Aldrich (Schnelldorf, Germany). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI-1640) medium, penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Lonza (Verviers, Belgium). ELISA kits for interleukin-6 (IL-6), macrophage-inflammatory protein-3α (MIP3A), inflammatory chemokine monocyte chemotactic protein-1 (MCP-1), were purchased from R&D Systems (Abingdon, UK).

To measure the NO production, nitrite concentration in the culture supernatant was determined using Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride in 5% H3PO4) (Cayman Chemical, Ann Arbor, Michigan, USA). 100 μl of cell culture supernatant and 100 μl Griess reagent were mixed and incubated for 10 min, to color development. The absorption was estimated at 540 nm, using a Glomax Multireader spectrophotometer (Promega, Madison, WI, USA). Nitrite standard (Cayman Chemical, Ann Arbor, Michigan, USA) was used to generate a standard curve for quantification. Results were obtained from three independent experiment measurements.