Figure 4

Cilia formation in BBEC cultures over time. Bronchial epithelial cell cultures were grown for the indicated number of days at an ALI (relative to the establishment of the ALI) before fixation. The BBEC cultures were subsequently processed to assess ciliation using (A) H&E staining of histological sections (arrowheads indicate ciliated cells), (B) immunofluorescence staining (cilia - green; F-actin - red; nuclei - blue) and (C) SEM. Representative images are shown of BBECs grown for 0, 12, 21 and 41 days post-ALI (see also Figs S1, S5 and S6). Quantitative analysis (using ImageJ) of ciliation of BBEC cultures fixed at three-day intervals ranging from day −3 to day 42 post-ALI was performed (D) by counting the number of ciliated cells per field of view in H&E-stained sections (see Fig. S1) and (E) using fluorescence intensity thresholding of immunostained cultures (see Fig. S5). In (D) for each insert, ciliated cells were counted in each of five randomised 400x fields of view evenly distributed across the strand. In (E) ciliation was quantified by measuring the area above a fluorescence intensity threshold in ImageJ; for each insert, five regions evenly distributed across the sample were measured. For all of the above quantifications, three inserts were analysed per time-point and the data represents the mean +/−standard deviation from tissue derived from three different animals (n = 9). Statistical significance was tested using an Ordinary one-way ANOVA: ns = not significant.