Figure 2
Optical characterisation of in vitro cultures. Representative brightfield images of the apical surface of epithelial monocultures (A), healthy fibroblast co-cultures (B), and diseased fibroblast co-cultures (C). Quantification of apical surface contrast (D), as a proxy for mucus coverage, for epithelial monocultures and co-cultures. Scale bar = 1 mm, N = 3 representative images for each condition. Haematoxylin and eosin stain (left) and immunofluorescent stain (right) of representative histological slices of epithelial monocultures (E), healthy fibroblast co-cultures (F), and diseased fibroblast co-cultures (G). Scale bars = 50 µm, insets = × 1.5 magnification. Purple arrow heads indicate goblet cells and black arrow heads indicate cilia projections. Anti-E-cadherin can be visualised in green (Cell Signaling Technology, 0.25 μg/ml), anti-MUC5B in yellow (Abcam, 0.025 μg/ml), anti-MUC5AC in red (ThermoFisher, 0.0015 μg/ml) and DAPI in blue (Akoya Biosciences). Quantification of cell morphology for representative slices including epithelial height (H), nucleus size (I), roundness (J), and aspect ratio (K). Statistical analysis was performed using a one-way ANOVA with Tukey’s multiple comparisons (****, P = < 0.0001; *** = 0.0001), N = 160 for NHBEs, N = 65 for NHBE + NHLF and N = 75 for NHBE + DHLF. For epithelial height N = 50. Data are presented as the mean ± SD. NHBE = Normal Human Bronchial Epithelial cell; NHLF = Normal Human Lung Fibroblast; DHLF = Diseased Human Lung Fibroblasts.
