title: A method for evaluating the use of fluorescent dyes to track proliferation in cell lines by dye dilution.
abstract: Labeling nonquiescent cells with carboxyfluorescein succinimidyl ester (CFSE)-like dyes gives rise to a population width exceeding the threshold for resolving division peaks by flow cytometry. Width is a function of biological heterogeneity plus extrinsic and intrinsic error sources associated with the measurement process. Optimal cytometer performance minimizes extrinsic error, but reducing intrinsic error to the point of facilitating peak resolution requires careful fluorochrome selection and fluorescent cell sorting. In this study, we labeled the Jurkat and A549 cell lines with CFSE, CellTraceViolet (CTV), and eFluor 670 proliferation dye (EPD) to test if we could resolve division peaks in culture after reducing the labeled input widths by cell sorting. Reanalysis of the sorted populations to ascertain the level of reduction achieved always led to widths exceeding the gated limits due to the contribution of errors. Measuring detector-specific extrinsic error by sorting uniform fluorescent particles with similar spectral properties to the tracking dyes allowed us to determine the intrinsic error for each dye and cell type using a simple mathematical approach. We found that cell intrinsic error ultimately dictated whether we could resolve division peaks, and that as this increased, the required sort gate width to resolve any division peaks decreased to the point whereby issues with yield made A549 unsuitable for this approach. Finally, attempts to improve yields by setting two concurrent sort gates on the fluorescence distribution enriched for cells in different stages of the cell cycle that had nonequivalent proliferative properties in culture and thus should be practiced with caution.
authors.affiliation: FACS Laboratory, London Research Institute, Cancer Research UK, London, WC2A 3LY, United Kingdom.
authors.forename: Julfa
authors.initials: J
authors.lastname: Begum
authors.forename: William
authors.initials: W
authors.lastname: Day
authors.forename: Carl
authors.initials: C
authors.lastname: Henderson
authors.forename: Sukhveer
authors.initials: S
authors.lastname: Purewal
authors.forename: Joana
authors.initials: J
authors.lastname: Cerveira
authors.forename: Huw
authors.initials: H
authors.lastname: Summers
authors.forename: Paul
authors.initials: P
authors.lastname: Rees
authors.forename: Derek
authors.initials: D
authors.lastname: Davies
authors.forename: Andrew
authors.initials: A
authors.lastname: Filby
mesh_terms: D049109:Cell Proliferation; D004395:Dye Dilution Technique; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D019169:Jurkat Cells; D015203:Reproducibility of Results; D013194:Staining and Labeling; D013388:Succinimides / Q000737:chemistry
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides
keywords: CFSE; cancer; cell lines; cell proliferation; dye dilution; eFluor proliferation dye; imaging flow cytometry; immunology; stem cells, cell trace violet