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.