Primary normal human bronchial epithelial cells (NHBE) cultured at air liquid interface with or without macrophage cells (+/- Mac) derived from primary peripheral blood mononuclear cells

The purpose is to obtain samples for mRNA analysis in primary normal human bronchial epithelial cells cultured with or without macrophage cells (+/- Mac) derived from primary peripheral blood monnumclear cells Influenza A/Vietnam/1203/2004 (H5N1, VN1203) or influenza A/California/04/2009 (H1N1, CA04) viral stocks were used to infect NHBE cells at a multiplicity of infection (MOI) of 1. Infections were allowed to progress for 1 hour. Next, 500,000 peripheral blood mononuclear cell (PBMC)-derived macrophages were place onto each apical transwell surface in a final volume of 500 ul to create co­ cultures. The co-cultures at a ratio of approximately 2 NHBE cells for every PBMC-derived macrophage were allowed to progress for an additional 11, 15 and 23 hours respectively (12, 16, 24 hours post-infection). An n=5 was used for each condition, which included infection with either H1N1, H5N1, or mock uninfected, of either NHBE-only monocultures or NHBE + PBMC-macrophage co-cultures. In total this infection scheme represented a total of 90 samples for microarray analysis. At the specified time points, 1 ml of TRlzol RNA isolation reagent (Thermo Fischer, 15596-016) that lead to inactivation of samples.