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Updated: Jul 16, 2025
Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes
Published on: May 8, 2016
A novel method for isolation and flow cytometry analysis of intraepithelial lymphocytes from colon biopsies
K Lamers1, M A Steele1, L R Cangiano1
1Department of Animal Biosciences, University of Guelph, Guelph, ON, N1G 2W1, Canada.
Insights
This study presents a new method to analyze intestinal immune cells from calf colon biopsies. This technique allows for precise identification of gamma delta T cell subsets, improving understanding of gut health.
Area of Science:
- Veterinary Immunology
- Gastrointestinal Research
- Cellular Immunology
Background:
- Intestinal immune responses are crucial for animal health but are difficult to study directly.
- Current methods rely on indirect markers or tissue gene expression, limiting detailed analysis.
- A direct method to analyze intestinal immune cells is needed.
Purpose of the Study:
- To develop and validate a protocol for isolating and analyzing intestinal intraepithelial lymphocytes (IEL) from bovine colon biopsies.
- To enable precise characterization of immune cell populations, particularly gamma delta (γδ) T cells, within the intestine.
- To facilitate research into how management and nutrition impact intestinal immunity.
Main Methods:
- Collection of bovine colon biopsy samples at different ages (d 2, 28, 42).
- Isolation of IEL using a Hanks' balanced salt solution and digestion solution.
- Flow cytometry analysis of IEL, including density gradient separation for improved sample quality.
- Identification of γδ T cells and their subsets using specific fluorescent antibodies.
Main Results:
- A validated protocol for isolating and analyzing IEL from colon biopsies was established.
- Flow cytometry enabled precise identification and quantification of lymphocyte populations.
- The proportion of different γδ T cell subsets within intestinal IEL was evaluated over time.
Conclusions:
- This novel technique allows for direct characterization of intestinal immune function in cattle.
- The method provides a valuable tool for researchers studying gut health and immune responses.
- It will aid in understanding the effects of various strategies on intestinal immunity.
Abstract:
Investigating the immune responses of the intestine in response to different insults is predominantly limited to indirect methods such as circulating markers of intestinal health or gene expression from dissections. We describe here a validated protocol for the isolation and subsequent flow cytometry analysis of intestinal intraepithelial lymphocytes (IEL) from colonic biopsy samples. Colon biopsy samples were collected with endoscopy forceps from Holstein dairy bull calves at d 2, 28, and 42 of life. The biopsies were put into an isolation solution of Hanks' balanced salt solution, and fetal bovine serum followed by digestion solution. The solution was filtered and the flow-through, containing IEL, was stained with fluorescent antibodies for flow cytometry analysis. Density gradient separation of the isolate yielded higher viability and cleaner samples for flow cytometry analysis. Anti-bovine γ chain of the T cell receptor was used to identify populations of gamma delta (γδ) T cells via flow cytometry. In addition, γδ T cell subsets were identified using an anti-bovine antibody against the coreceptor workshop cluster 1. This method allowed for the precise identification of lymphocyte populations and evaluation of the proportion of different subsets of γδ T cells from intestinal IEL over time. The technique described here will allow the research community to characterize intestinal immune function over time and improve our understanding of how different management and nutritional strategies affect intestinal health.
