Monocyte Isolation and Activation Assay

Buffy coats from healthy donors were obtained after written informed consent (Sanquin Blood Bank, Nijmegen, the Netherlands). Human peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation over Ficoll-Paque (GE Healthcare). Percoll isolation of monocytes was performed as previously described as yielding a level of T cell contamination, measured by fluorescence-activated cell sorting of only 5% [13 (link), 17 (link)]. Purified monocytes were cultured in RPMI 1640 Dutch-modified culture medium (RPMI medium, Invitrogen) supplemented with 50 μg/mL gentamicin (Centraform), 2 mmol/L Glutamax (Invitrogen), 1 mmol/L pyruvate (Invitrogen), and 10% pooled human serum. Stimuli and inhibitors used were Escherichia coli lipopolysaccharide (LPS; serotype 055:B5, Sigma-Aldrich, 10 ng/mL), Pam3Cys (EMC Microcollections, L2000, 10 μg/mL), 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO, Sigma-Aldrich), and 2-deoxy-d-glucose (2-DG, Sigma-Aldrich). Low-density lipoprotein was isolated from pooled human serum by ultracentrifugation and oxidized by incubating with 20 μmol CuSO₄/L for 16 h at 37 °C followed by dialysis, as previously described [4 (link)].