Early induction of TNF-α expression followed by late induction of mannose receptor (MR) in 3D cultures. Cells were exposed to 0.5 μg/ml (0.38 μg/cm2) of particulates. After 1, 3, 7, 10 and 14 days, cells were incubated with Brefeldin A for 6 hrs, fixed in situ and co-immunostained for TNF-α (red), MR (green), and counterstained with DAPI (blue nuclear fluorescence). A three-day exposure to asbestos or MWCNTs resulted in an increase in TNF-α expression. Prolonged exposure to all nanomaterials resulted in an increase in MR expression. Samples were analyzed by confocal microscopy using the appropriate filters. Photographs are representative of Z-stacks of three individual samples. Magnification: 400×.