Figure 3

Silica nanoparticles are internalized by alveolar macrophages and colocalize with alveolar epithelial cells. C57Bl/6j mice were instilled with FITC-SiO₂ NPs (5 mg/kg;100 μg/mice). A) 5 h later, mice were sacrificed and cells present in BALs were analysed by flow cytometry after labelling with specific antibodies directed against alveolar macrophages markers (CD11c, F4/80). Panel B) shows that 15 ± 2% (n = 4, mice) of the cells were FITC positive. Among them, over 95% were CD11c + and 85% CD11c + F4/80+ (panel C). The FACs analysis from one representative BAL from a total of 4 is shown. In D) BAL cells were cytospined and stained with anti-mouse CD45 antibody followed by Alexa 664 Fluor (A664) coupled secondary antibody (red) to confirm internalisation of FITC-SiO₂ NPs (white arrows). E) Cryo-sections of lung tissues recovered from mice 24 h after instillation of FITC- SiO₂ NPs (green) were stained with anti-proSPC antibody (a marker of alveolar type II epithelial cells) followed by A664 coupled secondary antibody (red). White arrows indicate co-localisation of NPs and alveolar epithelial cells. White scale bar = 100 μm or 20 μm (upper and lower panel, respectively). Nuclei were stained with DAPI (blue). Fluorescence of stained cells and tissues were analysed under structured illumination (ApoTome system). Superposed images from FITC, A664, DAPI and differential interferential contrast (D, lower panel) filters are shown. The ‘A’ within the figures indicates the alveolar spaces.