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. 2013 Aug;49(2):190–196. doi: 10.1165/rcmb.2012-0485OC

Figure 3.

Figure 3.

New BCs can be generated from adult tracheal epithelium in vitro. (A) Gα-gustducin (red) immunoreactive BCs in tracheal epithelial cell culture on air–liquid interface (ALI) Day 7. Cultured BCs have multiple processes like their in vivo counterparts. (B) BCs in cultures produced from TRPM5-GFP (green) transgenic mice were immunoreactive for Gα-gustducin (red) and expressed GFP on ALI Day 7. (C) Gα-gustducin (green) immunoreactive BCs were negative for the ciliated cell marker acetylated tubulin (ACT, magenta) on ALI Day 7. (D) A double-labeled cell with a BrdU (green) and Gα-gustducin (red) double-immunoreactive BC (arrow). BrdU also labeled other cells in culture (ALI Day 14). (E) Several doublet Gα-gustducin (red) immunoreactive BCs, suggestive of daughter cells, were observed culture on ALI Day 1. (F) Double-labeled image shows a Gα-gustducin (red) immunoreactive doublet, where both nuclei are labeled with BrdU (green), on ALI Day 14. Counterstains with 4′6-diamidino-2-phenylindole are shown in blue. Scale bars = 10 μm. (G) Tracheal epithelial cell density (red line) and the fraction of BCs (green line) over time in ALI cultures. Points depict the mean ± SEM (n = 3) for each time. (H) Fraction of cultured BCs labeled when dosed with BrdU, either during the proliferation period (Pro 3) or on ALI Day 0. *P < 0.001, according to t test. Bars depict the mean ± SEM (n = 3) for each point.