2.2. Cell culture

Human monocyte-derived dendritic cells (mDC) were cultured from monocytes obtained from apheresis transfusion cones (National transfusion service U.K.) by ficoll density centrifugation and positive CD14 selection using micro-beads (Miltenyi, U.K.) to achieve monocyte cultures that were >90% pure. Monocytes were cultured for 6-days in RPMI1640 containing 5% FCS, 20 ng/ml GM-CSF (Gibco, U.K.) and 4 ng/ml IL-4 (BD Pharmingen, U.K.). Murine bone marrow derived macrophages (BMDM) were isolated from the femurs of littermate wild type (cybb^+/+ or pkr^+/+), gp91 phox deficient (cybb^−/−) or PKR deficient (pkr^−/−) C57BL6 mice and cultured for 7-days in RPMI1640 containing 10% FCS and supplemented with 5% L929 conditioned media. Wild type (gcn2^+/+) or GCN2 deficient (gcn2^−/−) mouse embryonic fibroblasts (MEFs) were cultured in DMEM containing 10% FCS and supplemented with 55 μM β-mercaptoethanol.