Confocal microscopy images (×10 mag) of DC migration
36 h
after allergen stimulation in 3D tricoculture. The upper scaffold
contained the Calu-3 epithelial layer (A), a middle layer to which
DCs were seeded (B), and a lower scaffold containing MRC-5 (C). DCs
were prestained with Hoescht nuclear stain (blue) and Calu-3 cells
were poststained with pancytokeratin (green). In single culture, DCs
remained on the middle scaffold where they had been inoculated. In
the triculture, most DCs migrated from middle scaffold (B) to upper
scaffold (A). Upon treatment of the triculture with house dust mite
extract (HDM) (10 μg/mL) and lipopolysaccharide (LPS) (100 ng/mL),
DCs appeared to primarily migrate to the apical surface of the epithelial
layer, whereas in unstimulated samples they appear to be mainly localized
in the basal region of the epithelial layer (A). Experiments were
performed in duplicates.