Tovar Elizabeth A, Sheridan Rachael, Essenburg Curt J, Dischinger Patrick S, Arumugam Menusha, Callaghan Megan E, Graveel Carrie R, Steensma Matthew R
Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, USA.
Flow Cytometry Core, Van Andel Research Institute, Grand Rapids, Michigan, USA.
Bio Protoc. 2020 Nov 20;10(22):e3818. doi: 10.21769/BioProtoc.3818.
With the advent of CRISPR-Cas and the ability to easily modify the genome of diverse organisms, rat models are being increasingly developed to interrogate the genetic events underlying mammary development and tumorigenesis. Protocols for the isolation and characterization of mammary epithelial cell subpopulations have been thoroughly developed for mouse and human tissues, yet there is an increasing need for rat-specific protocols. To date, there are no standard protocols for isolating rat mammary epithelial subpopulations. Analyzing changes in the rat mammary hierarchy will help us elucidate the molecular events in breast cancer, the cells of origin for breast cancer subtypes, and the impact of the tumor microenvironment. Here we describe several methods developed for 1) rat mammary epithelial cell isolation; 2) rat mammary fibroblast isolation; 3) culturing rat mammary epithelial cells; and characterization of rat mammary cells by 4) flow cytometric analysis; and 5) immunofluorescence. Cells derived from this protocol can be used for many purposes, including RNAseq, drug studies, functional assays, gene/protein expression analyses, and image analysis.
随着CRISPR-Cas技术的出现以及轻松修改各种生物体基因组的能力，越来越多的大鼠模型被开发出来，用于探究乳腺发育和肿瘤发生背后的遗传事件。针对小鼠和人类组织，已经全面开发了乳腺上皮细胞亚群的分离和表征方案，但对大鼠特异性方案的需求也日益增加。迄今为止，尚无分离大鼠乳腺上皮亚群的标准方案。分析大鼠乳腺层次结构的变化将有助于我们阐明乳腺癌中的分子事件、乳腺癌亚型的起源细胞以及肿瘤微环境的影响。在此，我们描述了几种已开发的方法，用于：1）大鼠乳腺上皮细胞分离；2）大鼠乳腺成纤维细胞分离；3）培养大鼠乳腺上皮细胞；以及通过4）流式细胞术分析和5）免疫荧光对大鼠乳腺细胞进行表征。源自该方案的细胞可用于多种目的，包括RNA测序、药物研究、功能测定、基因/蛋白质表达分析和图像分析。
