Macs human cd14 microbeads

Fresh PBMCs from two healthy donors were separated from whole blood samples using Ficoll-Paque^TM. CD14+ monocytes were selected using MACS human CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), pre-separation filters (Miltenyi), and LS columns (Miltenyi) following the manufacturer’s recommendations. To induce M0 macrophages, isolated CD14+ monocytes were seeded onto FBS-coated 24-well plates at a density of 1 × 10⁵ cells/cm². Seeded cells were cultured for 7 days in RPMI 1640 media supplemented with 20% FBS and 100 ng/ml M-CSF (BioLegend, San Diego, CA, USA). On day 7, M-CSF-containing medium was removed, and appropriate stimulating media containing 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) and 20 ng/ml IFN-γ (BioLegend) for M1 induction or 20 ng/ml IL-4 (BioLegend) and 20 ng/ml IL-10 (BioLegend) for M2 induction were supplied. After an additional 48 h of culture, cells were collected by gentle scraping and then taken for fluorescence-activated cell sorting (FACS) analysis or scRNA-seq. The morphological changes that occurred during differentiation were observed every 2 days under a microscope. A fraction of the cells was triple-stained with PE/Cy7 anti-human CD14 antibody (BioLegend), FITC anti-human CD80 antibody (BioLegend), and PE anti-human CD163 antibody (BioLegend) and analyzed by FACSVerse and FACSuite v1.2 (BD Biosciences).

Monocytes used for nuclear protein extraction were isolated from apheresis blood and for ChIP and bisulfite sequencing monocytes were isolated from PBMCs derived from functional cohort by positive selection using MACS Human CD14 Microbeads (Miltenyi Biotec) according to the instructions recommended by manufacturer. Neutrophils used for the bisulfite sequencing were isolated by Ficoll-Paque density gradient centrifugation. The neutrophils were isolated from the layer immediately above erythrocyte layer and lyzing the erythrocyte with erythrocyte lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) for 10 min at room temperature on a roller mixer. The neutrophils were washed once with cold RMPI1640 supplemented with 10% fetal bovine serum.

Monocytes from individuals heterozygous for rs257174 were purified from PBMCs by positive selection using MACS human CD14 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions. Monocytes were plated at 0.5 × 10⁶ cells/well in 24-well tissue culture plates and then incubated with or without cell culture-grade ATP disodium salt hydrate (Sigma-Aldrich) in RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml of penicillin and 100 μg/ml of streptomycin. For the inhibition of ATP the incubation was carried out in the presence of 10 μM of the purinergic receptor inhibitor A-438079 hydrochloride (Tocris Bioscience). Monocytes were incubated for 2 h before harvesting using cell scrapers. The treated monocytes were stored in TRIzol (Invitrogen Life Technologies) at -80°C until further analysis.