Figure 4.

Novel mAb clone E5D10 enriches for both Lgr5⁺and Bmi1⁺intestinal stem cell populations. (A) High-magnification immunofluorescent image of acetone-fixed frozen murine small intestinal tissue stained with mAb E5D10 (red). White dashed lines represent the epithelial-mesenchymal boundary. Fluorescent images were captured on a Zeiss Observer Z1 microscope with ApoTome. Scale bar: 25 μm. Data are representative of N = 5 mice. (B) FACS plot of mouse small intestinal epithelial cells stained with mAb E5D10. Inset: Isotype staining control. N = 6 independent experiments, N = 12 mice. (C) qRT-PCR analysis of gene expression from FACS-isolated E5D10^lo (dark gray) and E5D10^hi (dashed lines) cells relative to the parent live epithelial population. Representative data from technical replicates. Gene expression analyses were performed on N = 3 independent experiments from N = 6 mice. Data are means ± SEM of triplicate fold change. (D) FACS plot of Lgr5^GFP (left) and Lgr5^GFP-hi cells (right) or (E) Bmi1^GFP cells from reporter mice stained with mAb E5D10. N = 6 independent experiments, N = 12 mice per genotype. (F) qRT-PCR analysis of stem cell and Paneth marker genes from an isolated E5D10^lo cell population relative to the parent live epithelial cells. Representative data from technical replicates. Gene expression analyses were performed on N = 3 independent experiments from N = 6 mice. Data are means ± SEM of triplicate fold change. FACS analyses were performed on a BD Influx cell sorter at the OHSU Flow Cytometry Core Facility. SSC, side-scattered light.