A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells

Scott T. Magness, Brent J. Puthoff, Mary Ann Crissey, James Dunn, Susan J. Henning, Courtney Houchen, John S. Kaddis, Calvin J. Kuo, Linheng Li, John Lynch, Martin G. Martin, Randal May, Joyce C. Niland, Barbara Olack, Dajun Qian, Matthias Stelzner, John R. Swain, Fengchao Wang, Jiafang Wang, Xinwei WangKelley Yan, Jian Yu, Melissa H. Wong

    Research output: Contribution to journalArticle

    17 Scopus citations

    Abstract

    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 FACSisolating 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.

    Original languageEnglish (US)
    Pages (from-to)G542-G551
    JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
    Volume305
    Issue number8
    DOIs
    StatePublished - Oct 15 2013

    Keywords

    • Epithelial dissociation
    • FACS
    • Intestinal epithelium
    • Intestinal stem cell

    ASJC Scopus subject areas

    • Physiology
    • Hepatology
    • Gastroenterology
    • Physiology (medical)

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  • Cite this

    Magness, S. T., Puthoff, B. J., Crissey, M. A., Dunn, J., Henning, S. J., Houchen, C., Kaddis, J. S., Kuo, C. J., Li, L., Lynch, J., Martin, M. G., May, R., Niland, J. C., Olack, B., Qian, D., Stelzner, M., Swain, J. R., Wang, F., Wang, J., ... Wong, M. H. (2013). A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells. American Journal of Physiology - Gastrointestinal and Liver Physiology, 305(8), G542-G551. https://doi.org/10.1152/ajpgi.00481.2012