Preparation of human monocyte-derived macrophages
Monocytes were isolated from the peripheral blood by Ficoll/Histopaque gradient centrifugation. Cells were washed once in phosphate-buffered saline (PBS) and adjusted to a cell of 2 × 10^6 ml in RPMI 1640 medium (Biochrom) with 2mM glutamine, 10% heat-inactivated fetal calf serum (FCS, Sigma), 2% HEPES (Biochrom), 1% penicillin/streptomycin (Gibco) and 1mM sodium pyruvate (Sigma-Aldrich). 500µl was seeded into each well of 24- well tissue culture. After 1 h of incubation, cells were washed twice with PBS to remove non-adherent cells. Cells were further incubated with medium containing 25 nM granulocyte macrophage colony stimulating factor (PeproTech), which was additionally added every second day. The cells were further polarized into M1 macrophages with lipopolysaccharide (LPS, 100 ng/μl) one day before start of the experiment. After 7 days of incubation, cells were used for experiments.