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Updated: Aug 8, 2026
Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches
Published on: March 17, 2013
Confocal microscopical analysis of epithelial cell heterogeneity in mouse Peyer's patches
P S James1, C Rossetti, M W Smith
1AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, UK.
Insights
Researchers directly compared cyanine dye fluorescence and alkaline phosphatase activity in mouse Peyer's patch cells. This study identifies antigen-transporting M cells in living tissue using membrane potential measurements.
Area of Science:
- Immunology
- Cell Biology
- Microscopy
Background:
- Antigen-transporting M cells in the Peyer's patch follicle-associated epithelium are crucial for mucosal immunity.
- Previous identification of M cells relied on low alkaline phosphatase activity in fixed tissue.
- The functional state of M cells in living tissue remains largely uncharacterized.
Purpose of the Study:
- To test the hypothesis that antigen-transporting M cells possess a low membrane potential.
- To develop a method for identifying M cells in living tissue.
- To correlate membrane potential with alkaline phosphatase activity in Peyer's patch epithelial cells.
Main Methods:
- Confocal microscopy was used to compare cyanine dye fluorescence (membrane potential) and alkaline phosphatase activity.
- Living mouse Peyer's patch tissue was equilibrated with the membrane potential-sensitive dye DIOC5(3).
- Fixed tissue was incubated with naphthol AS-BI phosphate and Fast Red TR to visualize alkaline phosphatase activity.
Main Results:
- A linear correlation was observed between membrane potential and alkaline phosphatase activity.
- Cells with low alkaline phosphatase activity in fixed tissue exhibited low DIOC5(3) fluorescence in living tissue.
- This indicates that antigen-transporting M cells can be recognized by their low membrane potential in vivo.
Conclusions:
- Antigen-transporting M cells in the Peyer's patch exhibit a low membrane potential.
- DIOC5(3) fluorescence provides a method to identify M cells in living tissue.
- This technique facilitates the study of cell surface-antigen interactions in real-time within the Peyer's patch.
Abstract:
Cyanine dye fluorescence and alkaline phosphatase activities have been compared directly by confocal microscopy in a wide variety of cells present in the follicle-associated epithelium of the mouse Peyer's patch to test the hypothesis that antigen-transporting M cells have a low membrane potential. In order to make these comparisons it was first necessary to equilibrate living tissue with the membrane potential sensitive dye DIOC5(3), fix with glutaraldehyde and then incubate the fixed tissue with naphthol AS-BI phosphate, a substrate which is hydrolysed by alkaline phosphatase present in the luminal membrane of these epithelial cells. Naphthol AS-BI produced by this reaction is then coupled to Fast Red TR diazonium salt at the site of hydrolysis. Selecting the 488 nm wavelength of the argon laser source then allows one to measure alkaline phosphatase activities as Fast Red absorbance and membrane potentials by DIOC5(3) fluorescence. Results obtained show a linear correlation between membrane potential and alkaline phosphatase activity. Relative lack of alkaline phosphatase activity, determined in fixed tissue, has been used previously to identify antigen-transporting M cells (Smith et al., 1987). The present work shows that it is now possible to recognize these cells in living tissue by measurement of DIOC5(3) fluorescence. The possible importance of this finding in providing a way to study cell surface-antigen interactions taking place in living tissue is discussed.
