Isolation of human PBMC
Buffy coats were obtained from healthy blood donors in compliance with the Helsinki declaration and with the approval of the ethics committee of the University of Luebeck. The buffy coats were separated over a Ficoll-Hypaque (Biochrom, Berlin, Germany) gradient [54,55]. Following isolation, PBMC were cultured at a final concentration of 3 × 106 cells/ml (for ELISA measurements) or 5 × 106/ml (for gel shifts) or 1 × 106 cells/ml (for flow cytometry) in RPMI 1640 medium (BioWhittaker, Heidelberg, Germany, LPS-free), containing 10% heat-inactivated fetal calf serum (FCS, low-LPS, Invitrogen, Karlsruhe, Germany), 1% penicillin (10000 U/ml), 1% streptomycin (10000 μg/ml), and 1% 200 mM L-glutamine (BioWhittaker).