Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches
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Document Type
Video
Publication Date
3-17-2013
DOI
10.3791/50167
Abstract
Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and Drosophila Larval IHC, a JoVE Science Education video explaining more about about the context of immunostaining" >immunostaining For flow cytometry, PPs are mechanically dissociated and then filtered through 70 μm membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of >2.5 x 106 cells with >90% cell viability For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome Tissue sections (5-12 μm) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.
Recommended Citation
De Jesus, Magdia, "Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches" (2013). Biomedical Sciences Faculty Scholarship. 5.
https://scholarsarchive.library.albany.edu/bms_fac_scholar/5
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This work is made available under the Scholars Archive Terms of Use.
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Publisher Acknowledgment
Copyright Journal of Visualized Experiments, 2013
De Jesus, M., Ahlawat, S., & Mantis, N. (n.d). Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches. Jove-Journal Of Visualized Experiments, (73)