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. 2018 Mar 10;6(1):79–96. doi: 10.1016/j.jcmgh.2018.02.007

Figure 2.

Figure 2

Novel mAb clone F5C12 enriches for Lgr5+intestinal stem cells. (A) Immunofluorescent image of acetone-fixed frozen murine small intestinal tissue stained with mAb F5C12 (red). (B) Immunofluorescent image of Lgr5-GFP reporter mouse small intestinal tissue stained with antibodies against GFP (green) and mAb F5C12 (red). Data are representative of N = 5 mice. White dashed lines represent the epithelial-mesenchymal boundary. Fluorescent images were captured on a Zeiss Observer Z1 microscope with ApoTome. Scale bar: 25 μm. (C) FACS plot of intestinal epithelial cells stained with mAb F5C12. Inset: Isotype staining control. (D) FACS plot of total Lgr5GFP cells (left) and Lgr5GFP-hi (right) or (E) Bmi1GFP cells from reporter mice stained with mAb F5C12. N = 6 independent experiments, N = 12 mice per genotype. Data are means ± SEM. FACS analyses were performed on a BD Influx cell sorter at the OHSU Flow Cytometry Core Facility. (F) qRT-PCR analysis of crypt and cell lineage marker expression from FACS-isolated F5C12+ intestinal epithelial cells relative to the unsorted live epithelial parent population. (G) qRT-PCR analysis of stem cell and Paneth marker gene expression from an isolated F5C12+ cell population relative to crypt-epithelial cells. qRT-PCR data shown are from 1 representative independent experiment, N = 2 mice. Data are representative of N = 3 independent experiments from N = 6 total mice. Data are means ± SEM from triplicate technical replicates. (H) FACS plot of intestinal epithelial cells from Lgr5-GFP reporter mice stained with mAb F5C12. (I) Analyses of seeding and growth efficiency in standard medium plus CHIR-99021 and valproic acid. Images of resultant enteroids grown from singly isolated cells from the populations boxed in red shown in panel H. Asterisk denotes autofluorescence in lumen. (J) Quantification of enteroid growth efficiency from single cells of each population from panel H. Data shown are from 1 independent culture experiment and are representative of N = 3 independent experiments from N = 6 total mice. Data are means ± SEM. Images were captured on an Olympus ×81 inverted microscope. Scale bar: 200 μm. SSC, side-scattered light.