Heparinized blood samples (400 mL) were fivefold diluted in 24well plates in 1.6 mL RPMI 1640 medium (BioWhittaker, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (QB perbio, UK), 2 mmol/L L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin. For stimulation, a mixture of 10 ng/mL PMA (Sigma, France), 1 mg/mL ionomycin (Sigma, France) and 1 mg/mL LPS from Escherichia coli O111:B4 (Sigma, France) was added to the diluted blood. For mock stimulation, a volume of PBS equal to the volume of stimulation reagents was added to the diluted blood. After incubation at 37uC for 24 h, culture supernatants were collected by centrifugation at 450 g for 20 min and stored at 220uC before use.