Abstract
The intestinal epithelial layer forms tubular invaginations into the underlying connective tissue of the lamina propria. These structures, termed crypts, are the basic functional unit of the intestine. Colon crypts and the surrounding lamina propria house different cell types, including epithelial cells, stem cells, enterocytes, goblet cells, as well as cells of the innate and adaptive immune systems (Clevers, 2013; Mowat and Agace, 2014). Here we describe a technique for the isolation of mouse intestinal crypt cells as well as their characterization by flow cytometry analysis (FACS) (Del Reino et al., 2012).
Keywords: Flow cytometry, Colon, Intestinal, Crypt cell, Mouse
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Sequential images of colon isolation. Pictures were acquired sequentially as described in the text. Figure 2. Light microscope images of mouse colon crypts. 20 μl of a suspension containing the crypts were placed directly on glass objective slides together with a glass coverslip. Images were acquired with a 10x and a 40x objective and a digital camera attached to the microscope. A higher magnification of the field in the dotted squared is shown in the right panel. Crypts are in single or cluster elongated columnar and spherical cells. Figure 3. Characterisation of CD45+ cells in mouse colon. Colon crypt cells from mouse were stained with anti-CD45, -CD4, CD8, -Ly6G and -F4/80 antibodies and the percentage of positive cells analysed by flow cytometry. Representative profiles are shown. Other FACS analysis and quantification image examples are published in Cancer Research (Del Reino et al., 2014). For details, refer to Figure 5B.
Recipes
Acknowledgments
This work was supported by The Spanish Ministry of Economy and Competiveness (MINECO) (SAF2013-45331-R) and La Marató TV3 Foundation (82031).
References
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