Whole blood stimulation and flow cytometry
Levels of STAT1 and phosphorylated (p)STAT1 were evaluated by flow cytometry as previously described [17]. Briefly, heparinized whole blood samples were stimulated with IFN-γ; 400 UI/mL; Inmukin, Horizon Pharma) for 15 min at 37°C. Cells were permeabilized by 1-hour incubation with ice-cold methanol and stained with antibodies against CD14, STAT1, and pSTAT1 (supplementary Table 1), for 1h at 4°C in the dark. Stained cells were fixed (1% paraformaldehyde, Sigma), and acquired (BD LSRII FORTESSA™). Data was analyzed with the FlowJo (Tristar Treestar, Ashland, OR, USA) software package.