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The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids.

Publication ,  Journal Article
Rothschild, DE; Srinivasan, T; Aponte-Santiago, LA; Shen, X; Allen, IC
Published in: J Vis Exp
May 18, 2016

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

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Published In

J Vis Exp

DOI

EISSN

1940-087X

Publication Date

May 18, 2016

Issue

111

Location

United States

Related Subject Headings

  • Tissue Culture Techniques
  • Receptors, Pattern Recognition
  • Organoids
  • Mice
  • Intestine, Small
  • Animals
  • 3101 Biochemistry and cell biology
  • 1702 Cognitive Sciences
  • 1701 Psychology
  • 0601 Biochemistry and Cell Biology
 

Citation

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Rothschild, D. E., Srinivasan, T., Aponte-Santiago, L. A., Shen, X., & Allen, I. C. (2016). The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids. J Vis Exp, (111). https://doi.org/10.3791/54033
Rothschild, Daniel E., Tara Srinivasan, Linette A. Aponte-Santiago, Xiling Shen, and Irving C. Allen. “The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids.J Vis Exp, no. 111 (May 18, 2016). https://doi.org/10.3791/54033.
Rothschild DE, Srinivasan T, Aponte-Santiago LA, Shen X, Allen IC. The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids. J Vis Exp. 2016 May 18;(111).
Rothschild, Daniel E., et al. “The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids.J Vis Exp, no. 111, May 2016. Pubmed, doi:10.3791/54033.
Rothschild DE, Srinivasan T, Aponte-Santiago LA, Shen X, Allen IC. The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids. J Vis Exp. 2016 May 18;(111).

Published In

J Vis Exp

DOI

EISSN

1940-087X

Publication Date

May 18, 2016

Issue

111

Location

United States

Related Subject Headings

  • Tissue Culture Techniques
  • Receptors, Pattern Recognition
  • Organoids
  • Mice
  • Intestine, Small
  • Animals
  • 3101 Biochemistry and cell biology
  • 1702 Cognitive Sciences
  • 1701 Psychology
  • 0601 Biochemistry and Cell Biology