Differentiation capacity of immortalized BCi-NS1.1 cells over long-term culture. A. Morphology of ALI day 28 of BCi-NS1.1 cells, passage 30 demonstrating a multilayer ciliated epithelium, hematoxylin and eosin staining B. Immunofluorescent staining of ALI day 28 of BCi-NS1.1 cells, passage 30, for KRT5 (basal cell, red), β-tubulin IV (ciliated cell, green) and nuclei (blue). A, B. Bar = 20 μm. C. Quantification of differentiation of ALI day 28 cross-sections of BCi-NS1.1 cells. The number of positive ciliated and secretory cells was scored for BCi-NS1.1 cells for n = 10 independent experiments from cells between passage 6 to 43. The data is presented for each cell type as percentage relative to the total number of cells. In addition, the average ± the standard error of n = 10 independent experiments is shown. D. Comparison of the differentiation capacity of BCi-NS1 cells to that of primary airway BC. Primary BC were cultured under identical differentiation-inducing conditions on ALI to that of BCi-NS1.1 cells and quantified in an identical manner. The number of positive ciliated and secretory cells was scored for primary BC and presented for each cell type as percentage relative to the total number of cells. The average ± the standard error of n = 5 independent experiments is shown and compared to the average data for BCi-NS1.1 described in panel C. Statistics were calculated by a 2-tailed Student’s t test.