**2. Materials and Methods**

This study was conducted according to good clinical practice guidelines and in line with the principles outlined in the Helsinki Declaration of the World Medical Association. Ethics approval was obtained from the Human Clinical Research and Ethics Committee of the University Hospital Virgen Macarena (PI00082017) and all subjects gave written, informed consent.

### *2.1. Human Postprandial Study and TRL Isolation*

Six volunteers, aged 25 to 35 years, non-smokers, with no medical history of disease known, nor any abnormality of hematological or biochemical parameters, were recruited in Clinical Biochemistry Unit at the University Hospital Virgen Macarena (UHVM, Seville). After an overnight fasting period of 12 h, all of them were given, over three different occasions, an oral fat emulsion containing cow's milk cream (meal rich in SFAs), refined olive oil (meal rich in MUFAs) or refined olive oil plus a dose of omega-3 long-chain PUFAs, which consisted of 920 mg of EPA and 760 mg of DHA (a meal rich in PUFAs). They also consumed the same test meal without fat as a control meal. Oral fat emulsions were prepared according to the method described by our Patent WO/2014/191597. They consisted of water, sucrose, fat (50 g/m2 body surface area), emulsifier, and flavoring. At fasting (0 min) and after the ingestion of the meals within 10 min, blood samples were collected each hour into K3EDTA-containing Vacutainer tubes (Becton Dickinson, NJ, USA) over 6 h. Postprandial TRLs were isolated, pooled, and dialyzed

against cold phosphate-buffered saline (PBS) [16]. TRLs were then immediately stored at −80 ◦C. Lipid oxidizability of postprandial TRL was checked (Thiobarbituric acid reactive substances level) during isolation and storage, but oxidation of lipids was not detected. TRLs were tested for lipopolysaccharide (LPS) contamination using the Pierce LAL Chromogenic Endotoxin Quantification kit (Thermo Scientific, Madrid, Spain). LPS contamination was always <0.2 EU/mL. TG concentration in postprandial TRLs was determined by colorimetric assay kit TG GPO-POD (Bioscience Medical, Madrid, Spain).

#### *2.2. Fat and TRL Fatty Acid Composition*

The fatty acid composition of cow's milk cream, refined olive oil, and refined olive oil plus omega-3 long-chain PUFAs was determined, in triplicate from the same lot, by the method described in EEC/796/2002 [17] using a gas chromatography system (HP-5890, Hewlett-Packard, Waldbronn, Germany) equipped with flame ionization detector and a SP-2380 capillary column (Supelco, 30 m × 0.32 mm) packed with cyanopropylsiloxane (0.25 μm) (Supplementary Materials Table S1). The initial column temperature was 165 ◦C, which was held for 10 min, then programmed from 165 ◦C to 200 ◦C at 1.5 ◦C/min. Injector and detector temperature were 250 ◦C, with the carrier gas H2.

For fatty acid composition in postprandial TRLs (named TRL-SFAs from cow's milk cream, TRL-MUFAs from refined olive oil, and TRL-PUFAs from refined olive oil plus omega-3 long-chain PUFAs), aliquots of 100 μL were lyophilized [18]. A solution composed of methanol: toluene: dimethoxypropane: sulphuric acid (16.5:5:1:1) and heptane was added on the lyophilized residue. After shaking and incubating the mixture at 80 ◦C for 1 h, the upper phase was transferred to another vial and dried with a stream of N2 gas. The resulting extract was dissolved in heptane and the FA methyl esters were analyzed into a gas chromatography system as described above (Table 1).


**Table 1.** Fatty acid composition of postprandial TRLs.

Data are expressed as mean ± SD, *n* = 18. TRL: triglyceride-rich lipoprotein; SFAs:saturated fatty acids; MUFAs: monounsaturated fatty acids; PUFAs: polyunsaturated fatty acids.

#### *2.3. Monocyte Isolation*

The same six volunteers who took part as donors of postprandial TRLs participated as donors of monocytes. After an overnight fasting period of 12 h, peripheral blood samples were drawn from a large antecubital vein and collected into K3EDTA-containing tubes (BD). Peripheral blood mononuclear cells (MNCs) were isolated by centrifugation over a Ficoll–Histopaque (Sigma, Madrid, Spain) gradient [19]. Monocytes were isolated from peripheral blood MNCs using anti-CD14 microbeads and LS columns on a midiMACS system (MiltenyiBiotec, Madrid, Spain). Monocyte (CD14+) purity was routinely >90% by flow cytometry analysis (FACSCanto II flow cytometer and FACSDiva software, BD) and cell

viability >95% by trypan blue exclusion (Sigma). The monocytes were seeded in 24-well culture plates at a density of 1 <sup>×</sup> 106 cells/mL and cultured in ultra-low attachment flasks in RPMI 1640 medium supplemented with L-glutamine, penicillin, streptomycin, and 10% heat-inactivated fetal bovine serum (complete culture medium).

#### *2.4. MonocyteDerived Dendritic Cell Maturation and Activation*

Monocytes were seeded in 24-well plates (1 <sup>×</sup> 106 cells/well) and induced to differentiate for 6 days in the presence of human recombinant GM-CSF (50 ng/mL) and IL-4 (20 ng/mL) to obtain moDCs. Degree of differentiation of the resulting population was determined for CD123 antigen using anti-human CD123 monoclonal antibody (Miltenyi Biotec) by flow cytometry analysis (more than 95% of cells were positive for CD123) [20–22]. Complete culture medium was replaced every 2 days with fresh medium and the cytokines. To study the effect of TRLs on moDC differentiation, monocytes were treated for 6 days with TRL-SFAs, TRL-MUFAs, or TRL-PUFAs at 100 μg TG/mL in presence of GM-CSF and IL-4.

## *2.5. Monocyte-Derived Dendritic Cell Viability*

For cell viability, monocytes were seeded in 96-well plates (1 <sup>×</sup> 105 cells/well) and differentiated into moDCs as indicated above. At days 1, 2, 4, and 6, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sigma) was added to cells for 2 h until a purple precipitate was visible. MTT–formazan crystals were then solubilized with dimethyl sulfoxide (DMSO) (Sigma) and measured with a microplate reader at 570 nm corrected to 650 nm. Cell survival was expressed as the percentage of absorbance compared with that of the control, non-treated cells.

### *2.6. Triglyceride Quantification*

Cellular lipids were extracted using hexane/isopropanol (3:2, *v*/*v*). The supernatant was obtained after centrifugation at 500× *g* for 5 min. The TG content was measured using the assay kits GPO/PAP (Axiom Diagnostics, Burstadt and Worms, Germany). To determine the protein content, cells were sonicated in radioimmunoprecipitation assay (RIPA) buffer, and the lysate was measured using the Bradford protein assay (Bio-Rad Laboratories, Madrid, Spain).
