Rpmi 1640 cell medium

Roswell Park Memorial Institute-1640 (RPMI-1640) cell medium, trypsin-EDTA, and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Sigma-Aldrich.
LM36R was maintained in RPMI-1640 supplemented with 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA, USA), 4 mM glutamine, 100 µg/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich) in an atmosphere of 37 °C with 5% CO₂. Media were changed every 2 days.

Peripheral blood mononuclear cells (PBMCs) were collected from three to five (indicated in each figure legend) healthy human blood donors by venipuncture in EDTA-coated vacutainer tubes (Sarstedt). Due to the fact that blood was only obtained from the authors, according to our local ethics committee (University of Freiburg Ethics Committee), under the relevant national and local regulations, ethical approval and informed consent was not needed. PBMCs were separated from other blood components by Ficoll-Plaque (GE Healthcare Life Sciences) density gradient centrifugation and resuspended in MACs buffer containing anti-CD14 microbeads (Miltenyi Biotec). The isolation was performed via positive selection using the MS MACs Column (Miltenyi Biotec) and the MiniMACs magnet (Miltenyi Biotec) according to the manufacturer’s protocol. The CD14+ monocytes were counted and seeded at a density of 50,000 cells/ml in RPMI-1640 cell medium (Sigma Aldrich) containing 10% FBS (Bio Chrome) and PenStrep (Life Technologies Corporation). The CD14+ monocytes were treated with maturation factors GM-CSF (10 ng/mL, Peprotech) or M-CSF (25 ng/mL, Peprotech) to induce M1 or M2 macrophages, while M0 macrophages were left untreated. The cell suspensions were placed in T25 flasks (Greiner Bio-One) for two days.

Collagenase (Type VIII), dimethyl sulfoxide (DMSO), E-Toxate™ reagent from Limulus Polyphemus, fetal bovine serum (FBS), hyaluronidase, monosodium urate crystals (MSU), and RPMI-1640 cell medium were purchased from Sigma‐Aldrich Co. (Milan, Italy). Flow cytometry fixation and permeabilization buffer kit I, proteome profiler mouse cytokine array kit and recombinant mouse Gal-9 were purchased from R&D System (Milan, Italy). FACS buffer and conjugated antibodies from BioLegend (London, UK). Ficoll-Paque Plus (endotoxin tested, ≤ 0.12 EU/ml) was obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Unless otherwise stated, all the other reagents were from BioCell (Milan, Italy).