Gibco s serum free aim 5 medium

Heparinized leukocyte-enriched buffy coats were obtained from healthy blood donors, and peripheral blood mononuclear cells (PBMCs) were separated from buffy coats by Ficoll-Paque Plus (Biosciences) gradient centrifugation. Monocytes were purified from PBMCs by positive selection using immunomagnetic cell separation and anti-CD14-conjugated microbeads (Miltenyi Biotec), according to the manufacturer’s instructions. After separation on a VarioMACS magnet, 96–99% of the cells were shown to be CD14⁺ monocytes.
Isolated monocytes were cultured for 5 days in 6-well tissue culture plates at a density of 2.0 × 10⁶ cells/ml in Gibco’s serum-free AIM-V medium (Thermo Fischer Scientific) supplemented with 50 ng/ml M-CSF (PeproTech). In order to acquire the M1 and M2 types, cells were stimulated on the fifth day of differentiation for 24 h with lipopolysaccharide (50 ng/ml ultrapure LPS, InvivoGen), IFNγ (20 ng/ml, PeproTech) to M1 and IL-4 (20 ng/ml, PeproTech), IL-10 (20 ng/ml, PeproTech) and TGFß (20 ng/ml, PeproTech) to M2 phenotype. For the differentiation of TAM-like cells, isolated monocytes were cultured for 5 days in 6-well tissue culture plates at a density of 2.0 × 10⁶ cells/ml in Thp-1 supernatant supplemented with IL-4 (20 ng/ml), IL-10 (20 ng/ml) and TGFß (20 ng/ml). On the fifth day, TAM-like cells were treated again with Thp-1 supernatant for 24 h.

Peripheral blood mononuclear cells (PBMCs) were separated by a standard density gradient centrifugation with Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden). Monocytes were purified from PBMCs by positive selection using immunomagnetic cell separation and anti-CD14 microbeads, according to the manufacturer’s instruction (Miltenyi Biotec, Bergisch Gladbach, Germany). After separation on a VarioMACS magnet, 96–99% of the cells were shown to be CD14⁺ monocytes, as measured by flow cytometry. Isolated monocytes were cultured for 2 days in 12-well tissue culture plates at a density of 5.0 × 10⁵ cells/ml in Gibco’s serum-free AIM-V medium (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 80 ng/ml GM-CSF (Gentaur Molecular Products, Brussels, Belgium) and 100 ng/ml IL-4 (PeproTech EC, London, UK). The cells were differentiated in the presence or absence of 1 nM ATRA followed by a 75-min incubation period with or without 1 μM BMS614 specific RARα-antagonist at 37°C atmosphere containing 5% CO₂.

To generate THP-1 supernatant, cells were cultured at a density of 2 × 10⁵ cells/ml in Gibco’s serum-free AIM-V medium (Thermo Fischer Scientific) for 2 days and the supernatant was collected at 1500 rpm for 5 min.