SL
Scott W Lowe
  • Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, USA
Research fields
  • Stem cell
Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells
Authors:  Kevin P. O’Rourke, Sarah Ackerman, Lukas E Dow and Scott W Lowe, date: 02/20/2016, view: 28203, Q&A: 6
In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro.
Immunofluorescent Staining of Mouse Intestinal Stem Cells
Authors:  Kevin P. O’Rourke, Lukas E Dow and Scott W Lowe, date: 02/20/2016, view: 32738, Q&A: 1
Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, permeabilize, stain and mount intestinal organoids for analysis by immunofluorescent confocal microscopy.


Figure 1. A schematic depicting a crypt-villus forming organoid, and visualization of Paneth cells by immunofluorescence staining. Left: Small intestinal organoids grow as crypt-villus structures that contain all of the multiple differentiated lineages of the intestine. Right: Immunofluorescent staining can be used to visualize individual cell types in the organoid. Here paneth cells are visualized by staining for lysozyme (“Lyso,” Green), which reveals Paneth cells located at crypt bases. F-Actin (Red) reveals crypt structure at the apical surface of the epithelium, and DAPI (Blue) reveals cell nuclei. Scale bar is 25 μm.
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