PBMCs were incubated with magnetic microbeads (130-091-153, Monocyte Isolation Kit II, Miltenyi Biotec, Bergisch Gladbach, Germany) in accordance with the
manufacturer’s protocol, and final isolation of monocytes was achieved using a magnetic cell sorter (AutoMACS, Miltenyi Biotec, Germany). PBMCs were differentiated into dendritic cells using an established protocol (Rogler et al., 1998); monocytes were cultivated in flasks for 1 week under optimal conditions (37 °C, 5% CO2, 95% relative humidity [RH]) with 5 ng/mL IL-4 and 50 ng/mL granulocyte–macrophage colony-stimulating factor (GM-CSF). As described above for ivDCs, peripheral blood monocytes were differentiated into macrophages based on the this website established protocol cited, with monocytes being cultivated in Teflon bags for 1 week under optimal conditions (37 °C, 5% CO2, 95% RH). Differentiated macrophages were detached from the Teflon bags by incubation at 4 °C. The monocytic/macrophage-like THP-1 cell line was cultivated in Roswell Park Memorial Institute medium containing 10% fetal calf serum (FCS), supplemented with penicillin (100 U/mL) see more and streptomycin (100 μg/mL) under standard conditions (37 °C, 5% CO2, 95% RH). Human epithelial colorectal adenocarcinoma (Caco-2) cells were
grown in high glucose Dulbecco’s Modified Eagle’s Medium containing 10% FCS, supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) under recommended conditions (37 °C, 10% CO2, 95% RH). For both cell lines, passaging was carried out according to the guidelines
of the American Type Culture Collection (ATCC, 2012). The influence of retinoids on the LPS-induced cytokine response of ivDCs, ivMACs and THP-1 cells was evaluated in each cell type using the same experimental methodology. Primary cells were adjusted to a density of 1 × 106 cells/mL and plated onto 96-well plates (100 μL/well). THP-1 Urease cells were incubated in six-well plates at a density of 7 × 105 cells/mL. Retinoids were added to the medium (0.01, 0.1, 1.0 and 5.0 μg/mL) for ivDCs, ivMACs and THP-1 cells, and pre-incubated for 1 h prior to stimulation with LPS (to a final concentration of 100 ng/ml) for a further 48 h at 37 °C. Incubation medium was collected and processed for cytokine analysis (Rules-Based Medicine, Austin, Texas, USA) using Human Cytokine MAP A 1.0® array. Levels of IL-15 were below the detection limit of the assay and were excluded from the analysis. For studies in ivDCs, cytokine response data shown are based on at least six (LPS-induced) and at least four (no LPS) independently performed experiments, each corresponding to a different donor. In ivMACs, these data (both LPS-induced and no LPS) were each based on at least four independently performed experiments, each corresponding to a different donor. Data shown for cytokine response in THP-1 cells are based on three independent experiments.