For stimulation assays, 4 × 105 monocytes were exposed to ultrapure LPS (20 ng/ml) (catalog # tlrl-pelps, Invivogen) for either 2 or 24 hours. Alternatively, monocytes were exposed to IFNγ (catalog # 285-IF, R&D Systems) at a concentration of 20 ng/ml for 24 hours. The number of treated samples per individual depended on the total number of cells purified, and the priority on sample accrual was collation of IFNγ-treated followed by 24-hour LPS and then 2-hour LPS, respectively. Experiments were terminated simultaneously, ensuring identical incubation periods for all samples. A subset of samples was kept in the incubator without stimulation throughout this period to ascertain incubator effects. All experiments were completed within 48 hours of blood sample collection.
