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Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue

Nature Protocols volume 2, pages 2307–2311 (2007)Cite this article

Abstract

Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by quality control experiments to obtain cells that can be used for further investigations. After different steps of digestion of the tissue using collagenase, DNase and dispase, the resulting cells are purified using Percoll gradient. The success of the isolation can be analyzed by cell viability test (Trypan Blue exclusion test) and by flow cytometric analysis to assess apoptosis. Finally, the isolated cells can be used for further investigations like comparative studies of mRNA expression, cell-proliferation assay or protein analysis. This protocol can be completed within 6–7 h.

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Figure 1
Figure 2: Demonstration of the procedure to handle the colon during preparation.
Figure 3: Isolation of LPMCs from B/6 mice as described in PROCEDURE.
Figure 4: Immunofluorescence of cryosections of colon from B/6 mice performed using the Tyramide signal amplification Cy3 system (Perkin-Elmer) and a fluorescence microscope (Olympus).
Figure 5: Isolation of LPMCs as described, and consequent staining of cells with propidium iodide (PI) and Annexin V to discriminate between dead and apoptotic cells.

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References

  1. Schieferdecker, H.L., Ullrich, R., Hirseland, H. & Zeitz, M. T cell differentiation antigens on lymphocytes in the human intestinal lamina propria. J. Immunol. 149, 2816–2822 (1992).

    CAS  PubMed  Google Scholar 

  2. Breese, E., Braegger, C.P., Corrigan, C.J., Walker-Smith, J.A. & MacDonald, T.T. Interleukin-2- and interferon-gamma-secreting T cells in normal and diseased human intestinal mucosa. Immunology 78, 127–131 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Guy-Grand, D., Cuenod-Jabri, B., Malassis-Seris, M., Selz, F. & Vassalli, P. Complexity of the mouse gut T cell immune system: identification of two distinct natural killer T cell intraepithelial lineages. Eur. J. Immunol. 26, 2248–2256 (1996).

    Article  CAS  PubMed  Google Scholar 

  4. Bull, D.M. & Bookman, M.A. Isolation and functional characterization of human intestinal mucosal lymphoid cells. J. Clin. Invest. 59, 966–974 (1977).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Fiocchi, C., Battisto, J.R. & Farmer, R.G. Gut mucosal lymphocytes in inflammatory bowel disease: isolation and preliminary functional characterization. Dig. Dis. Sci. 24, 705–717 (1979).

    Article  CAS  PubMed  Google Scholar 

  6. Davies, M.D. & Parrott, D.M. Preparation and purification of lymphocytes from the epithelium and lamina propria of murine small intestine. Gut 22, 481–488 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Van der Heijden, P.J. & Stok, W. Improved procedure for the isolation of functionally active lymphoid cells from the murine intestine. J. Immunol. Methods 103, 161–167 (1987).

    Article  CAS  PubMed  Google Scholar 

  8. Neurath, M.F., Fuss, I., Kelsall, B.L., Stuber, E. & Strober, W. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J. Exp. Med. 182, 1281–1290 (1995).

    Article  CAS  PubMed  Google Scholar 

  9. Neurath, M.F. et al. Predominant pathogenic role of tumor necrosis factor in experimental colitis in mice. Eur. J. Immunol. 27, 1743–1750 (1997).

    Article  CAS  PubMed  Google Scholar 

  10. Wirtz, S., Becker, C., Blumberg, R., Galle, P.R. & Neurath, M.F. Treatment of T cell-dependent experimental colitis in SCID mice by local administration of an adenovirus expressing IL-18 antisense mRNA. J. Immunol. 168, 411–420 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Rogler, G. et al. Establishment of long-term primary cultures of human small and large intestinal epithelial cells. Lab. Invest. 78, 889–890 (1998).

    CAS  PubMed  Google Scholar 

  12. Riccardi, C. & Nicoletti, I. Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat. Protoc. 1, 1458–1461 (2006).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by SFB 548.

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Authors and Affiliations

  1. I. Med. Clinic, Immunological Laboratory, Johannes-Gutenberg University, Obere Zahlbacherstrasse 63, Mainz, 55131, Germany

    Benno Weigmann, Ingrid Tubbe, Daniel Seidel, Alex Nicolaev, Christoph Becker & Markus F Neurath

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  1. Benno Weigmann
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Correspondence to Benno Weigmann.

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Weigmann, B., Tubbe, I., Seidel, D. et al. Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue. Nat Protoc 2, 2307–2311 (2007). https://doi.org/10.1038/nprot.2007.315

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