A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells.
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.
Oregon Health & Science Univ., Dept. of Cell and Developmental Biology, Mailcode L215, RJH 5562, Portland, OR 97239. wongme@ohsu.edu.
Scott T
ST
Magness
Brent J
BJ
Puthoff
Mary Ann
MA
Crissey
James
J
Dunn
Susan J
SJ
Henning
Courtney
C
Houchen
John S
JS
Kaddis
Calvin J
CJ
Kuo
Linheng
L
Li
John
J
Lynch
Martin G
MG
Martin
Randal
R
May
Joyce C
JC
Niland
Barbara
B
Olack
Dajun
D
Qian
Matthias
M
Stelzner
John R
JR
Swain
Fengchao
F
Wang
Jiafang
J
Wang
Xinwei
X
Wang
Kelley
K
Yan
Jian
J
Yu
Melissa H
MH
Wong
D000818:Animals; D018929:Cell Culture Techniques; D002470:Cell Survival; D004847:Epithelial Cells / Q000502:physiology*; D005434:Flow Cytometry / Q000592:standards*; D005786:Gene Expression Regulation; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015588:Observer Variation; D013194:Staining and Labeling
D018960:Hyaluronan Receptors
FACS; epithelial dissociation; intestinal epithelium; intestinal stem cell