Human m csf

Buffy coats from healthy donors were obtained from Sanquin Blood Supply (Amsterdam, the Netherlands). Monocytes were isolated from buffy coat by density gradient centrifugation using Lymphoprep™ (Axis-Shield, Dundee, Scotland) followed by CD14⁺ selection via magnetic cell separation using MACS CD14 MicroBeads and separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany), as previously described.⁴¹ Alveolar-like MDMs were generated by differentiating CD4⁺ monocytes on tissue culture plates into macrophages in the presence of 50 ng/ml of human M-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany) for 6 days, followed by 24-h incubation in culture medium supplemented with 50 ng/ml IL10 (R&D System, Minneapolis, MN, USA). The resulting MDMs were then detached for stimulation using TrypLE Select (Gibco, Waltham, MA).

CD14+monocytes were isolated using positive magnetic separation with manufacturer instructions (Miltneyi) and were cultured in Iscove’s modified Dulbecco’s medium (Thermo Fisher) supplemented with 10% heat-inactivated FBS (Gibco), penicillin/streptomycin, and 50 ng/mL human M-CSF (Miltenyi Biotec). Monocytes were plated into 6-well plates (5×10⁵ cells) and incubated for 6 days with one medium change. Media from differentiated macrophages was replaced with fresh media containing drugs. The adherent macrophages were detached using macrophage detachment solution DXF (Sigma-Aldrich) for 40 min at +4°C, washed with flow cytometry running buffer, and blocked with Fc-blocking antibody (eBioscience) in flow cytometry running buffer for 10 min at room temperature (according to manufacturer instructions) before adding fluorescent antibodies. Macrophage median fluorescence intensity of surface CD163 and CD206 expression was analyzed on the NovoCyte Quanteon flow cytometer.