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Related Experiment Video

Updated: Oct 19, 2025

Isolating Lymphocytes from the Mouse Small Intestinal Immune System
11:28

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Published on: February 28, 2018

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Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue.

Chloe M Doyle1,2,3,4, Erica E Vine2,3,4, Kirstie M Bertram2,3,4

  • 1Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia.

Frontiers in Immunology
|September 27, 2021
PubMed
Summary

Researchers developed a new method to isolate viable, immature mononuclear phagocytes (MNP) from human intestines. This technique preserves cell surface markers for accurate flow cytometry analysis and functional studies of immune cells in the gut.

Keywords:
dendritic cells (DC)enzymatic digestionflow cytometryhuman tissueintestinemacrophage – cellmononuclear phagocyte cells (MNP)

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Last Updated: Oct 19, 2025

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Area of Science:

  • Immunology
  • Gastroenterology
  • Cell Biology

Background:

  • Human intestines host diverse mononuclear phagocytes (MNP) crucial for immune homeostasis.
  • Isolating viable MNP from intestinal tissue is challenging due to marker degradation and cell death during extraction.
  • Current methods often trigger MNP activation, altering their functional phenotype for analysis.

Purpose of the Study:

  • To develop an optimized method for isolating functionally immature intestinal MNP from human jejunum, ileum, and colon.
  • To enable high-parameter flow cytometry for comprehensive MNP subset identification.
  • To facilitate functional assays using sorted, viable intestinal MNP.

Main Methods:

  • Anatomical separation of intestinal layers (mucosa, submucosa) and lymphoid follicles.
  • Optimized enzymatic digestion protocols to yield viable, immature MNP.
  • Screening of antibody clones for high-parameter flow cytometry panels to identify MNP subsets (pDCs, cDC1, cDC2, DC3, monocytes, Mf1-4).
  • Novel method developed to correct for autofluorescence in tissue macrophages.

Main Results:

  • Successful isolation of viable, functionally immature intestinal mononuclear phagocytes.
  • Development of comprehensive flow cytometry panels capable of discriminating all known human intestinal MNP subsets.
  • Demonstrated utility of the method for sorting functional, immature dendritic cells for cytokine production assays.

Conclusions:

  • The described method provides a robust approach for isolating and analyzing diverse intestinal MNP subsets.
  • This technique preserves cellular integrity and function, enabling deeper understanding of MNP roles in intestinal immunity.
  • The optimized protocols and antibody panels are valuable tools for future research in gut immunology, disease, and immunotherapy.