Heat inactivated fetal calf serum

C57BL/6J mice were sacrificed by cervical dislocation and femur and tibia were isolated. Femur and tibia were flushed with PBS through a 70 μM filter to obtain a single cell suspension. After centrifuging, the cells were resuspended in complete medium: RPMI 1640 GlutaMAX (Gibco, Carlsbad, CA, USA), supplemented with 1% glutamine penicillin/streptomycin, 10% heat-inactivated fetal calf serum (Greiner Bio-One, Kremsmünster, Austria) and 15% L929 cell conditioned medium (LCM). Cells were cultured for 7 days to allow differentiation into bone marrow-derived macrophages (BMDMs).
Fatty acids PA (5 mM, Sigma-Aldrich, Saint Louis, MO, USA) and OA (50 mM, Sigma-Aldrich, Saint Louis, MO, USA) were dissolved in 96% ethanol. Next, the solutions were saponified by adding NaOH at a 5:1 (PA:NaOH) or 1:2 (OA:NaOH) ratio. The saponified solutions were evaporated under N2 at 37 °C. Boiling MQ was added to resuspend the saponified fatty acids. The fatty acids were dissolved in RPMI 1640 (Gibco, Carlsbad, CA, USA) containing 1% BSA (Sigma-Aldrich), resulting in solutions of 500 μM PA and 500 μM OA. BMDMs were stimulated with 50 μM PA, 50 μM OA and a combination of 50 μM PA and 50 μM OA for 16 h. After stimulation, BMDMs were washed with PBS and harvested for RNA isolation using TriReagent (Sigma-Aldrich, Saint Louis, MO, USA) or flow cytometry.

PBMCs were isolated by Ficoll density gradient centrifugation. The gradient was a commercially available Ficoll separation medium (Lymphoprep, Stem Cell Technologies, Cologne, Germany). PBMCs were resuspended in RPMI 1640 medium with Glutamax (Gibco Life Technologies, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal calf serum (Greiner Bio-One, Frickenhausen, Germany), of penicillin (100 U/ml) and streptomycin (1 µg/ml) as well as non-essential amino acids (NEAs) (diluted according to manufacturer’s instructions, MEM NEA) and sodium pyruvate (1 mM, all from Gibco Life Technologies, Schwerte, Germany).