Oregon Health & Science Univ., Dept. of Cell and Developmental Biology, Mailcode L215, RJH 5562, Portland, OR 97239.
Am J Physiol Gastrointest Liver Physiol. 2013 Oct 15;305(8):G542-51. doi: 10.1152/ajpgi.00481.2012. Epub 2013 Aug 8.
Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.
荧光激活细胞分选（FACS）是研究中分离不同肠上皮细胞群体所必需的工具。由于与 FACS 方法学相关的关键参数的报告不一致或缺乏报告，使得解释、比较和再现重要发现变得复杂。为了解决这个问题，设计了一项综合的多中心研究，以制定指导方针，限制实验和数据报告的变异性，并为研究之间的数据准确比较提供基础。建立了组织解离、细胞产量、细胞活力、FACS 和分选后纯度的常用方法和数据报告协议。七个中心通过 FACS 从鼠小肠分离特定隐窝基上皮群体（EpCAM+/CD44+）来测试标准化方法。对干细胞/祖细胞（Lgr5 和 Atoh1）和分化细胞谱系（溶菌酶、粘蛋白 2、嗜铬粒蛋白 A 和蔗糖异麦芽糖酶）的遗传生物标志物进行了检测，以评估靶群体和对照群体中的内中心和间中心变异性。对基因表达水平进行 Wilcoxon 秩和检验，显示生物重复之间的内中心变异性有限。主成分分析表明四个中心之间具有显著的中心间重现性。通过标准化细胞分离方法和数据报告要求收集的数据的分析，容易识别出方法学问题，表明标准报告参数有助于事后错误识别。这些结果表明，通过遵循常见的细胞分离方法和 FACS 门控策略，目标肠上皮细胞群体的 FACS 分离的复杂性可以在生物重复和不同机构之间具有高度的可重复性。这项研究可以被认为是进一步方法开发的基础，也是正在开发细胞分离专业知识以研究肠上皮生理学和病理生理学的研究人员的起点。
