1 m hepes

HT-1080 human fibrosarcoma cell line and MCF-7 human breast cancer cell line were kindly provided by Dr. Wei-Ting Chao. Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin and 1M HEPES for cell culture were purchased from Gibco, USA. Tetramyristoyl cardiolipin standard (14:0)₄ and tetralinoleoyl cardiolipin (18∶2)₄ extracted from bovine heart were purchased from Avanti Polar Lipids, USA.

Human keratinocyte cells (HaCaT, a spontaneously immortalized cell line) were obtained from Cell Lines Service GmbH (Eppelheim, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin, Hank’s Balanced Salt Solution (HBSS), MEM Non-Essential Amino Acids (NEAA) Solution (100×) and 1 M HEPES were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). For high-performance liquid chromatography (HPLC), all chemicals were of analytical reagent grade and were used as received. For mobile phase preparation, trifluoroacetic acid (TFA) and acetonitrile were purchased from Merck (Millipore, Darmstadt, Germany) and VWR (Barcelona, Spain), respectively. Dimethyl sulfoxide (DMSO) and the remaining reagents were purchased from Sigma-Aldrich (Steinheim, Germany). Apigenin and its monopotassium salt derivative (apigenin-K), which were 96.80% and 90.53% pure, respectively, were kindly provided by NUTRAFUR, SA—Frutarom Group (Alcantarilla, Murcia, Spain).

Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, 1 M HEPES and 0.5% trypsin-EDTA for cell culture were purchased from Gibco (Grand Island, NY, USA). Fatty-acid-free bovine serum albumin (BSA) was bought from Akron (Boca Raton, FL, USA). OA, LA, ALA, GLA and SDA were bought from Cayman (Ann Arbor, MI, USA). Primers were synthesized by ZGene Biotech Inc. (Taipei, Taiwan). iScript™ cDNA Synthesis kit and iQ™ SYBR® Green Supermix were acquired from Bio-Rad (Hercules, CA, USA). Invitrogen TRIzol reagent was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

The LUMINEX assay was performed according to the manufacture´s instructions MILLIPLEX MAP Kit (Human Cytokine / Chemokine 96-Well Plate Assay, 2009, Millipore Corporation, MA, USA). Briefly, eight different bead suspensions of antibodies, specifically directed against each of the eight cytokines (i.e. IL-1β, IL-2, IL-8, IL-10, IL-13, IL-17, TNF-α and IFN-γ, respectively), were irradiated with ultrasound and mixed in a solution. The 96-well filter plates were washed in RPMI (1640 medium without Phenol Red, Invitrogen, Gibco, NY, USA) with 1 M HEPES (Gibco, Paisley, Scotland, UK) at room temperature for 10 minutes while shaking. 25 μl standards, controls and samples were added in duplicates, and diluted 1:1 with RPMI with 1 M HEPES. The cytokine-bead solution was added to each well and the plate was incubated while shaking at 4°C overnight. The plates were analyzed on the Luminex 200 ^TM (Luminex Corporation, Austin, Texas, USA) using the Xponent software (Version 3.1, Software Solution for Luminex, Hoorn, The Netherlands). Samples with < 10 beads were excluded from analysis. Mean of duplicate samples represented the cytokine response.