2.3 In vitro culture of peripheral blood mononuclear cells
Blood samples were collected in tubes with lithium heparin (Vacutainer®) and diluted with an equal volume of warm PBS (Gibco, Invitrogen, Massachusetts). PBMCs were isolated by centrifuging at 800g for 20 min at 18-20oC on Biocoll separating solution (Biochrom AG, Germany). PBMCs were washed three times and the cell pellet was resuspended in complete medium [RPMI 1640 with HEPES 25 mM and L-Glutamine (Gibco, Life Technologies Ltd, UK), supplemented with 10ml/L Penicillin-Streptomycin USA, 50μl/L 1M β-mercaptoethanol, 20ml/L L-Glutamine plus MEM Vitamin, 20ml/L Non-essential Amino Acid, Sodium Pyruvate and 10% heat-inactivated FBS (all from Sigma-Aldrich, Germany)]. The suspension was seeded in a flat-bottom 48-well tissue plate (Corning Incorporated, Costar, New York), with 5 × 105 viable cells per well (500μL). PBMCs were cultured in duplicates either with complete medium alone (unstimulated control) or with one of the following stimulants: 4μg/ml Resiquimod (R848), 10μg/ml Zymosan (InvivoGen, France), 10μg/ml Phytohemagglutinin (PHA-M), 20μg/ml Polyinosinic–polycytidylic acid potassium salt (Poly I:C), (Sigma-Aldrich, Germany), at 37oC, 5% CO2. Cultures were harvested after 48 hours and, after centrifugation at 600g for 5 min, supernatants were stored at -80oC until analysis.