Rpmi 1640 media

Human peripheral blood mononuclear cells (hPBMCs) were isolated from buffy coats of healthy donors (Blodbanken i Oslo, Oslo Universitetessykehus) by Isopaque-Ficoll (Lymphoprep; Alere, A80102010) gradient centrifugation and incubated in flasks with 10 mL serum-free RPMI 1640 media (Biowest, L0496–500) for 90 minutes (min) for plastic adherence of monocytes before washing. Cells were then incubated for 6 days with RPMI 1640 media 10% FBS (Merck, F7524) and), 1% P/S (Merck, P4458) and 20 ng/mL macrophage colony stimulating factor (M-CSF; R&D Systems, 216-MC). For polarization, the cells were seeded in a 24-well plate and polarization was obtained towards the M1 or M2 phenotypes by incubation with RPMI 1640 supplemented with LPS (100 ng/mL) and IFNγ (20 ng/mL) for M1 polarization or IL-4 (20 ng/mL) for M2 polarization, for 18 h. The specific macrophage polarization was validated by measuring the mRNA expression of M1 and M2 markers in the cells by RT-qPCR. Cells isolated from a buffy coat were used in only one independent experiment, with multiple biological replicates. Two independent isolations from different buffy coats were performed.

BV2, murine microglial cells, were maintained at 37 °C and 5% CO2 in a humidified incubator. Cells were cultured in RPMI 1640 Media (Biowest) supplemented with 10% fetal bovine serum (Biosera) and 100 U/ml penicillin G and 100 mg/ml streptomycin sulfate. BV2 cells were plated on 24-well plates at a density of 3 × 10⁴ cells per well in RPMI complete medium. The next day, the cells were incubated in the presence or absence of Corinthian currant extract or a glucose/fructose mixture in serum-free medium for 24 and 48 h at 37 °C. The currant polar phenolic extract was used at concentrations 1 and 5 μg GAE/mL and the glucose/fructose mixture at concentrations 2.6 and 12.6 mM that match the glucose/fructose concentration of currant extract. The final methanol concentration in each well was 0.68% (v/v). The cell medium was collected after indicated treatments and supplemented with Complete Mini Protease Inhibitor Cocktail and 1 mM serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride. The cells were washed with ice-cold PBS and lysed at 4 °C in lysis buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.25% (w/v) deoxycholate, and Complete Mini Protease Inhibitor Cocktail). The protein concentration in cell lysates was determined using the Lowry assay (DC Protein Assay Kit, Bio-Rad).