Human primary macrophages were obtained from leukocyterich buffy coats from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). Monocytes were isolated as described previously [4] and differentiated into macrophages by maintenance in Macrophage-SFM medium (GIBCO) supplemented with 10 ng/ml GM-CSF (Biosource International) and antibiotics. After 7 days of culture, the resulting macrophages were used in experiments. Macrophages were infected with influenza A virus in complete Macrophage-SFM medium. The studied cells were lysed or fractionated into mitochondrial, cytoplasmic and nuclear fractions. Mitochondrial and cytoplasmic fractions were isolated by Qproteome Mitochondria Isolation Kit (Qiagen) and the cytoplasmic fractions were further purified with 2-D Clean-Up Kit (GE Healthcare). Nuclear fractions were isolated by Qproteome Nuclear Protein Isolation Kit (Qiagen) and the insoluble and soluble nuclear protein fractions were combined before analysis. About 1610 7 cells were used for all isolations. For secretome analyses, the cells grown in complete Macrophage-SFM medium were washed tree times with PBS after which the cells were stimulated in RPMI growth media supplemented with 1 mM HEPES, L-Glutamine and antibiotics (GIBCO). The growth media were collected and concentrated in Amicon Ultra centrifugal filter devices (Millipore Corporation, Billerica, MA).