Magnetic-activated cell sorting (MACS, Miltenyi) was performed to positively separate CD14+ monocytes from 20 million PBMCs, as per the manufacturer’s instructions. This is designed to provide a sample of ~99% purity. Flow cytometry was performed on a subset of samples with similar purities observed. Typical monocyte yields ranged from 1 × 106 to 10 × 106 monocytes per individual, of which 5 × 105 untreated cells were immediately suspended in RLT reagent (Qiagen) to form the naïve subset and snap-frozen for RNA extraction at a later date. All other monocytes were resuspended at a concentration of 1 × 106/ml in 400 μl of prewarmed RPMI 1640 complete medium, supplemented with 20% fetal calf serum, penicillin/streptomycin, and l-glutamine. Cells were rested overnight (16 hours) at 37°C, 5% CO2 in 5-ml nonadherent polypropylene cell culture tubes (BD Biosciences) before stimulation assays.
