Fig. 2

Lung epithelial cell isolation. Live single lung cells were stained for CD45, CD31, EpCAM, and MHCII. CD45⁻CD31⁻ cells were analyzed by cell sorting. a CD45⁻CD31⁻ cells were analyzed for EpCAM and MHCII expression to sort P1 cells (left). The sorted cells were reanalyzed (right). b CD45⁻CD31⁻ cells were analyzed for EpCAM expression and SSC (side scatter) to sort EpCAM⁺ cells (left). The sorted cells were reanalyzed (right). c Immunofluorescence analysis of cytospin preparations of sorted P1 cells. Almost all cells were positive for proSP-C expression. Scale bar, 20 μm. d FACS analysis of proSP-C expression in sorted P1 (left) or EpCAM⁺ cells (right). e The proportion of proSP-C⁺ cells analyzed by immunofluorescence among sorted P1 and EpCAM⁺ cells are shown (n = 3/group). *p < 0.05. f The proportion of proSP-C⁺ cells analyzed by FACS among sorted P1 and EpCAM⁺ cells are shown (n = 5/group). **p < 0.01. f mRNA expression in sorted P1 and EpCAM⁺ cells relative to that in whole lung cells (whole lung expression = 1). Note that sorted EpCAM⁺ cells have higher Foxj1 expression compared to sorted P1 cells, although both cells exhibit high Sftpc expression (n = 3/group). h Immunofluorescence analysis of cytospin preparations of sorted P2 cells demonstrating that almost all cells were positive for acetylated tubulin expression. Scale bars, 20 μm. i mRNA expression in sorted P2 cells relative to that in whole lung cells (whole lung expression = 1). Foxj1 is highly expressed (n = 3/group). j Immunofluorescence analysis of cytospin preparations of sorted P3 cells. Approximately half of the cells were positive for AQP5 expression. P3 cells were negative for SCGB1A1, proSP-C, acetylated tubulin, and T1α expression. (k) mRNA expression in sorted P3 cells relative to that of whole lung cells (whole lung expression = 1). Pdpn is highly expressed (n = 3/group). EpCAM, epithelial cell adhesion molecule; MHCII, major histocompatibility complex II; proSP-C, pro-surfactant protein C; ActTubulin, acetylated tubulin; SCGB1A1, secretoglobin 1A1; AQP5, aquaporin 5; DAPI, 4′,6-diamidino-2-phenylindole; PE, phycoerythrin; APC, allophycocyanin