Figure 1: Effect of culture medium and various lung fibroblast manipulations on the in vitro colony formation of lung epithelial stem cells Whole lung epithelial cells from Sftpc-GFP mice were co-cultured with lung fibroblasts in the in vitro colony forming assay in either “MTEC/Plus” or “lung epithelial” culture medium. Colony types were identified based on their morphology and GFP expression as detailed in Methods: C-type colonies express GFP in most cells, A and B types express less to no GFP. Yellow arrows point to some A/B colonies. A, B) Representative images of the effect of the difference in the medium used on colony size and type. C, D) Quantification of the growing colonies revealed that the MTEC/Plus medium was more efficient in supporting the colonies, as they induced the formation of a higher number of A/B colonies that were bigger in size and more complex, whereas the lung epithelial medium contained a higher number of colonies but smaller GFP+ alveolar Ctype colonies. Lung epi med: Lung epithelial medium.E) Percentage of live lung fibroblasts and mitomycin-C-treated feeder cells without freezing and after freezing and thawing. F) Whole lung epithelial cells from Sftpc-GFP mice co-cultured with equal numbers of live lung fibroblasts and mitomycin-C-treated feeder cells without (fresh) and after freezing and thawing. Yellow arrows point to some A/B colonies. G) Quantification of the number of growing colonies showed that freeze/thaw did not significantly reduce the ability of fibroblasts to support lung epithelial stem cell colony formation but significantly reduced such ability in mitomycin-C-treated feeder cells. Fibro: fibroblasts, F/T: freeze/thaw, MMC: mitomycin-C-treated feeder cells. Data represent results from three independent experiments. * p<0.05. Scale bars = 500 μm. A), B), and F) are merged images of bright field and the green fluorescence channel to detect which colonies are expressing GFP.