Lungs from Axin2CreERT2-TdTomato animals were harvested at 6–8 weeks of age and processed into a single cell suspension using dispase, collagenase I, and DNase as previously described36 (link),37 (link). EPCAM+ Axin2+ cells (TdTomato+) were identified via FACS sorting as previously described37 (link). The total AT2 population (Sftpc+ AT2s) was isolated from lungs of 6–8 week old SftpcCreERT2:R26REYFP animals 5 days after induction with 200μg/gm tamoxifen. EYFP+ cells were then isolated via FACS sorting as previously described37 (link). For sorting and quantification, the following antibodies were used: Pdpn-eFluor660 (eBioscience, Clone 8.1.1, 1:100) EpCAM-APC (eBioscience, Clone G8.8, 1:200), EpCAM-eFluor488 (eBioscience, Clone G8.8, 1:200), CD31-PeCy7 (eBioscience, Clone 390, 1:200), CD45-PeCy7 (eBioscience, Clone 30-F11, 1:200). Two anti-mouse Tm4sf1 antibodies were used to ensure specificity: Sheep anti-mouse Tm4sf1 (R&D systems, AF7514, 1:10) and Sheep IgG isotype control (R&D systems, 5-001-A, 1:10) with anti-Sheep 488 secondary (Abcam, ab150177, 1:50) or Rabbit anti-mouse Tm4sf1 (LS Biosciences, B7077, 1:25) and Rabbit IgG isotype control (LS Biosciences, LS-C109221, 1:25) with Donkey anti-rabbit 488 secondary (Life Technologies, A212016, 1:200).
Protocol detail
Isolation and Characterization of Lung Stem and Progenitor Cells
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Other organizations : University of Pennsylvania, Penn Center for AIDS Research
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Variable analysis
independent variables
- Age of animals (6-8 weeks)
- Presence and abundance of EPCAM+ Axin2+ cells (TdTomato+)
- Presence and abundance of total AT2 population (Sftpc+ AT2s)
- Mouse strains: Axin2CreERT2-TdTomato and Sftpc-CreERT2:R26R-EYFP
- Cell isolation and sorting protocols (dispase, collagenase I, DNase)
- Positive control: Sftpc+ AT2 cells isolated from Sftpc-CreERT2:R26R-EYFP mice
- Negative controls: Isotype controls for Tm4sf1 antibodies
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