Barrett Caitlyn W, Short Sarah P, Choksi Yash A, Williams Christopher S
Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, and Department of Cancer Biology, Vanderbilt University Medical School, Nashville, USA.
Bio Protoc. 2016 Jun 20;6(12). doi: 10.21769/BioProtoc.1837.
Small intestinal organoids, otherwise known as enteroids, have become an increasingly utilized model for intestinal biology as they recapitulate the various epithelial cells within the intestinal crypt (Mahe , 2013; Sato , 2009). Assessment of growth dynamics within these cultures is an important step to understanding how alterations in gene expression, treatment with protective and toxic agents, and genetic mutations alter properties essential for crypt growth and survival as well as the stem cell properties of the individual cells within the crypt. This protocol describes a method of visualization of proliferating cells within the crypt in three dimensions (Barrett , 2015). Whole-mount proliferation staining of enteroids using EdU incorporation enables the researcher to view all proliferating cells within the enteroid as opposed to obtaining growth information in thin slices as would be seen with embedding and sectioning, ensuring a true representation of proliferation from the stem cell compartment to the terminally differentiated cells of the crypt.
小肠类器官，也被称为肠小体，已成为肠道生物学中越来越常用的模型，因为它们能重现肠隐窝内的各种上皮细胞（马赫，2013年；佐藤，2009年）。评估这些培养物中的生长动态是理解基因表达改变、使用保护剂和毒性剂进行处理以及基因突变如何改变隐窝生长和存活所必需的特性以及隐窝内单个细胞的干细胞特性的重要一步。本方案描述了一种在三维空间中可视化隐窝内增殖细胞的方法（巴雷特，2015年）。使用EdU掺入对肠小体进行全组织增殖染色，使研究人员能够查看肠小体内所有增殖细胞，而不是像通过包埋和切片那样在薄片中获取生长信息，从而确保从干细胞区室到隐窝终末分化细胞的增殖情况得到真实呈现。
