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Exosomes release by dendritic cells: new quantification method using lasser scanning microscopy and image restoration
Titre de la conférence
15th Annual Congress of the European Respiratory Society (ERS)
Copenhagen, Denmark, September 17-21, 2005
Date de publication
European Respiratory Journal
Dendritic cells (DCs) realize a surveillance network in the lung. It has been shown that they could release small vesicles, called exosomes that might spread processed antigens or provide danger signals. The membranes of DCs were stained with either fluorescent probe DiO (green) or DiI (red). LPS-stimulated and unstimulated cells were co-cultured in a collagen matrix to trap the exosomes, and their release, size and incorporation were quantified after 6 h and 24 h. Cells were investigated by laser scanning microscopy and analyzed using the surpass module of the IMARIS software where confocal data could be segmented into individual objects allowing separation of the cell body from external objects (exosomes). When unstimulated cells were mixed with stimulated cells, in an early time (6h) unstimulated DCs incorporated exosomes into their cell membranes shown by the incorporation of red material, resulting in 25000 co-localized voxels compared to 7700 co-localized voxels in control cultures. In the same co-culture condition unstimulated cells released 21(SD 18) exosomes at 6 h and 442(SD 276) at 24 h, while unstimulated co-cultured cells released only 7(SD4) and 88(SD 64) exosomes respectively. Overall, DCs were able to release numerous exosomes in response to danger signals, and in an early stage they were able to integrate neighboring DCs' exosomes providing activation signals. Using the surpass module we were able to identify and quantify exosomes.
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