Fluorescence microscopy and flow cytometric analysis of Peyer's patches and intestinal immune cells

In recent years researchers have become more aware of the importance of the gut-associated immune system since it is in direct interaction with the entry site for virus, bacteria, and all type of food contaminants, including numerous toxins that can alter mucosal immunity. Peyer's patches (PP) are considered the inductive site for protein antigen presentation in the gut as well as the starting point for IgA B-cell differentiation. The IgA found in feces comes from IgA secreted by IgA lamina propria lymphocytes (LPL), and its presence is a sign of normal physiology, in that IgA plays a role in absorption and immune defense against gut-associated pathogens. Methods presented in this unit are intended to analyze PP and intestinal intraepithelial and LPL to determine whether the complexity of the mucosal-associated lymphoid tissue and its components have been altered by any form of external damage. The protocols explain how to isolate and culture isolated cells; how to stain and analyze; and also how to cryopreserve the gut.