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An enzymatic method for preparation of dissociated murine Peyer's patch cells enriched for macrophages
Abstract:
Enzyme (Dispase) dissociation of murine Peyer's patches (PP) 3 yields a cell population rich in T and B lymphocytes and containing 5-9% macrophages (MO) as defined by morphology and esterase staining. Cell yields by this method are 3-4 times greater than with conventional mechanical methods and viability of cells always exceeds 95%. The virtual absence of IgA plasma cells (less than 1%) indicates lack of contamination of the cell preparations with intestinal mucosal cells. Enzymatically dissociated PP cells proliferate in response to both T and B cell mitogens. Differences in patterns of mitogen responsiveness of sequentially obtained PP cell fractions are observed suggesting considerable functional heterogeneity of PP cells. Patterns of IgM and IgA synthesis by PP cells in response to the B cell polyclonal activator LPS differ considerably from spleen cells. The development of methodology for preparation of PP cells containing functionally intact T and B cells and enriched for MO should permit analysis of the cellular basis for functional specialization of PP.
Insights
Dispase enzyme dissociation of Peyer's patches (PP) yields viable T and B lymphocytes and macrophages (MO). This method improves cell yield and purity, enabling functional analysis of immune cells in PP.
Area of Science:
- Immunology
- Cell Biology
Background:
- Peyer's patches (PP) are crucial inductive sites for mucosal immunity.
- Conventional mechanical dissociation methods yield low cell numbers and viability.
- A need exists for improved methods to isolate functional immune cells from PP.
Purpose of the Study:
- To develop an enzymatic dissociation method for murine Peyer's patches (PP).
- To characterize the immune cell populations obtained using this method.
- To assess the functional integrity of isolated PP cells for further immunological studies.
Main Methods:
- Murine Peyer's patches were dissociated using the enzyme Dispase.
- Cell populations were analyzed for T and B lymphocytes and macrophages (MO) via morphology and esterase staining.
- Cell yield, viability, and purity were assessed, along with mitogen responsiveness and immunoglobulin synthesis.
Main Results:
- Dispase dissociation yielded 3-4 times more cells than mechanical methods, with >95% viability.
- Preparations were enriched in T and B lymphocytes and contained 5-9% macrophages (MO), with minimal IgA plasma cells.
- Enzymatically dissociated PP cells proliferated in response to T and B cell mitogens, showing functional heterogeneity.
Conclusions:
- Enzymatic dissociation with Dispase provides a superior method for obtaining viable, pure immune cells from murine Peyer's patches.
- This methodology facilitates the study of functional specialization within Peyer's patch immune cell populations.
- The isolated cells are suitable for analyzing the cellular basis of mucosal immune responses.