Statistical determination of threshold for cellular division in the CFSE-labeling assay.
The combination of flow cytometry and carboxyl fluorescent succinimidyl ester (CFSE) labeling techniques has been widely used in the study of cellular proliferation, including measurement of the percentage of proliferated cells and the number of cell divisions undergone by proliferated cells. However, the smallest numbers that represent true cell division rather than experimental variation are not known. To define a threshold that separates true proliferation from experimental variation, we performed a large number of replicate CFSE labeling experiments using polyclonal stimulation, obtained the percentages of proliferated cells using ModFit software, and then analyzed these data using several statistical methods. Our results indicate that the threshold of proliferation lies between 0.071% (95% confidence) and 0.114% (99% confidence) of total CFSE-labeled cells under our laboratory conditions. We offer our methods presented here for other investigators to calculate a threshold in their own CFSE-labeling experiments.
Liu
Dacheng
D
Department of Biostatistics and Computational Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, NY 14642, USA. dliu@rdg.boehringer-ingelheim.com
Yu
Jie
J
Chen
Huiyuan
H
Reichman
Richard
R
Wu
Hulin
H
Jin
Xia
X
D002455:Cell Division; D049109:Cell Proliferation; D003627:Data Interpretation, Statistical; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D013194:Staining and Labeling; D013388:Succinimides / Q000737:chemistry*
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides