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.