Ficoll paque

Blood was collected at participating sites in BD Vacutainer Hemogard Closure Plastic K2-Edta Tubes (BD Biosciences, CA). For the discovery cohort, an aliquot of fresh blood was used for imaging flow cytometry (see below). Peripheral blood mononuclear cells (PBMCs) were isolated within 4 h of collection using Ficoll-Paque density gradient centrifugation in Leucosep tubes (Greiner Bio-One, Austria). Cells were viably cryopreserved in RPMI 1640 (Life Technologies, Carlsbad, Calif) supplemented with 11.25% Human Serum Albumin (Gemini Bio-Products, West Sacramento, Calif)/10% dimethyl sulfoxide (Sigma-Aldrich, St Louis, Mo) and stored in liquid nitrogen until used or shipped to St John’s Institute of Dermatology. Prior to each experiment, cell count and viability were measured with Via1-Cassette on a NucleoCounter NC-200 (Chemometec, DK).

The PBMC fraction was obtained by density centrifugation of EDTA blood using a Ficoll-Paque gradient (Pharmacia Biotech, USA) or Leucosep tubes prefilled with Ficoll-Paque (Greiner, the Netherlands). After washing, the cells were re-suspended in RPMI 1640 Dutch modified (Gibco Invitrogen, USA) supplemented with 50 mg/L gentamycin (Centrafarm, the Netherlands), 2 mM L-glutamine, and 1 mM pyruvate (Gibco Invitrogen, USA). A volume of 100 µl containing 5×10⁵ mononuclear cells was added to round-bottom 96-well plates (Costar, Corning, The Netherlands) and incubated at 37°C and 5% CO₂ for both 4 h and 24 h with either 50 µl RPMI (medium control), C. burnetii NM 1×10⁷/ml, C. burnetii 3262 1×10⁶/ml, C. burnetii 3345937 3.2×10⁴/ml, C. burnetii 14160-001 1×10⁷/ml and E. coli LPS 10 ng/ml in the presence of 50 µl 5% naive goat serum (in house). Following incubation, the supernatant was harvested and stored at −20°C. Subsequently Trizol (Invitrogen, USA) was added to cell pellet and stored at −80°C till further RNA isolation.