Figure 5.

The combination of mAb clones E5D10 and F5C12 permits isolation of Lgr5^GFP-and Bmi1^GFP-enriched ISC populations. (A) Low-magnification and (B) high-magnification immunofluorescent images of acetone-fixed frozen mouse small intestinal tissue stained with mAbs E5D10 (green) and F5C12 (red). Data are representative of N = 5 mice. White dashed lines represent epithelial-mesenchymal boundary. Immunofluorescent images were captured on a Leica DMR upright microscope. Scale bar: 25 μm. (C) FACS plot of mouse intestinal epithelial cells stained with mAbs E5D10 and F5C12 and resultant cell populations of interest. Inset: Isotype staining control plot. N = 6 independent experiments, N = 12 mice. FACS analyses were performed on a BD Influx cell sorter at the OHSU Flow Cytometry Core Facility. (D) qRT-PCR gene expression analysis of FACS-isolated mAb populations E5D10^lo F5C12^neg (light blue) E5D10^lo F5C12⁺ (dark blue) and crypt (dashed lines) epithelial cells relative to villus epithelial cells. Representative data from technical replicates. Gene expression analyses were performed on N = 4 independent experiments from N = 8 mice. Data are means ± SEM of triplicate fold change. (E and F) Low-magnification (inset) and high-magnification brightfield images of resultant ex vivo 3D epithelial cultures derived from mAb cell populations (E) E5D10^lo F5C12^neg (F) E5D10^lo F5C12⁺ after 12 days of growth. (G) Quantification of culture growth phenotypes and growth efficiencies from single cells isolated from mAb sorted cell populations from N = 4 independent experiments, N = 8 mice. Culture images were captured on a Leica DMIRB inverted microscope. Scale bar: 200 μm.