PBMC Isolation and Stimulation Protocol

Peripheral blood mononuclear cells (PBMCs) were isolated healthy individuals (written informed consent was obtained from all subjects), as described earlier50 (link)51 (link). Briefly, PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare) and collecting the white interphase. Next, PBMCs were washed twice in cold PBS and concentrations were adjusted to 5 × 10⁶ cells/ml in RPMI-1640 Dutch Modified culture medium (RPMI supplemented with 2 mM l-glutamine, 1 mM pyruvate; GIBCO Invitrogen, Carlsbad, CA, USA). PBMC (5 × 10⁵), in a volume of 100 μl volume in round-bottomed 96-well plates (Greiner, Alphen a/d Rijn, The Netherlands), were incubated with either 100 μl of culture medium (negative control) or one of the following stimuli: B. quintana LPS and E. coli LPS (10 ng/ml).