Macrophage colony stimulating factor m csf

Peripheral blood mononuclear cells were isolated from a buffycoat (Sanquin blood supply, Amsterdam, the Netherlands) through density centrifugation using Lymphoprep™ (Axis‐Shield, Dundee, Scotland). Monocytes were then purified using human CD14 magnetic beads and MACS^® cell separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocytes were plated in 24‐well tissue culture plates at a density of 1×10⁶ cells/mL (500 μL per well) and differentiated to macrophages for 6 days in Iscove's Modified Dulbecco's Medium (Sigma‐Aldrich) supplemented with 2 mmol/L l‐glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL) and 10% fetal calf serum (All Gibco, Waltham, MA) in the presence of 50 ng/mL macrophage colony stimulating factor (MCSF) (Miltenyi Biotec, Bergisch Gladbach, Germany). On day 3, the medium was removed and substituted by fresh Iscove's Modified Dulbecco's Medium with 10% fetal calf serum and 50 ng/mL MCSF. On day 6, all medium was removed and replaced by Iscove's Modified Dulbecco's Medium with 10% fetal calf serum without MCSF and cells were activated for 18 hours with vehicle (DMSO), LPS (10 ng/mL, Sigma‐Aldrich), a liver X receptor (LXR) agonist (TO‐901317, 10 μmol/L, Sigma‐Aldrich), and LPS+LXR agonist. Total RNA was extracted from the cell lysate.

For in vitro macrophage induction, purified CD14+ monocytes were cultured in a complete RPMI medium (Life Technologies, Bleiswijk, The Netherlands) supplemented with 10% heat-inactivated calf serum (Merck Life Science, Madrid, Spain), 2 mM L-glutamine (Merck Life Science, Madrid, Spain), 1% antibiotic/antimycotic solution, and 100 ng/mL Macrophage Colony-Stimulating Factor (M-CSF) (Miltenyi Biotec, Bergisch Gladbach, Germany) for 8 days. Afterwards, M0 macrophages were polarized towards a M1 phenotype by incubating them with 120 ng/mL of IFNγ and 10 ng/mL of Lipopolysaccharides (LPS) (Merck Life Science, Madrid, Spain) for 48 h. For M2 phenotype, M0 macrophages were incubated with 2 µg/mL IL-4 (Peprotech, London, UK) for 48 h. MSCs were co-cultured with either M0, M1, or M2 macrophages at a ratio of 1:1. After 2 days macrophages were stained with the following antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany): anti-CD163-APC, anti-CD64-APC-Vio770, anti-CD209-PE-Vio770, anti-CD86-FITC, anti-MHC-I- PeVio770, anti-CD206 VioBlue, or with the corresponding isotype control antibodies. Data were acquired by flow cytometry using the MACSQuant^® analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Data were analyzed using MACSQuantify software (Miltenyi Biotec, Bergisch Gladbach, Germany) and FlowJo^TM v10.6.2 Software (BD Life Sciences, Ashland, OR, USA).

Human monocytes were isolated from leukocyte concentrate (buffy-coat) from healthy donors. Human PBMCs were obtained by the Ficoll-Paque density gradient centrifugation, and the CD14⁺ population was sorted using CD14 MicroBeads (Miltenyi) following the manufacturer's indications. Cells were plated at 2 × 10⁵ cells/cm² and cultured in an MLR medium supplemented with 20 ng/ml of macrophage colony-stimulating factor (M-CSF) (Miltenyi). After 7 days of culture, macrophages were activated with 100 ng/ml of lipopolysaccharides (LPS) and cultured alone, with naïve hAD-MSC or pretreated hAD-MSC at a ratio of 1 : 10 for 24 h. To assess macrophage polarization, cells were harvested using Versene (Gibco) and incubated with 100 μM of Fc block (BD Bioscience) following the manufacturer's indications. Then, cells were stained using a cell viability marker (Life Technologies) and antibodies against CD86 (clone 2331 Fun-1), CD163 (clone GHI/61) (BD Bioscience), CD68 (clone Y1/82A), CD206 (clone 15-2), and HLA-DR (clone L243) (BioLegend, San Diego, California, USA). Data were acquired with a BD FACSCanto and analyzed using the FlowJo software (v.10) (TreeStar Inc.).