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PLoS One. 2014 Jan 2;9(1):e84645. doi: 10.1371/journal.pone.0084645. eCollection 2014.

A simple FCM method to avoid misinterpretation in Saccharomyces cerevisiae cell cycle assessment between G0 and sub-G1.

Author information

1
INRA, UMR1083, Montpellier, France ; SupAgro Montpellier, UMR1083, Montpellier, France ; Universit Montpellier 1, UMR1083, Montpellier, France.

Abstract

Extensively developed for medical and clinical applications, flow cytometry is now being used for diverse applications in food microbiology. Most uses of flow cytometry for yeast cells are derived from methods for mammalian cells, but yeast cells can present specificities that must be taken into account for rigorous analysis of the data output to avoid any misinterpretation. We report an analysis of Saccharomyces cerevisiae cell cycle progression that highlights possible errors. The cell cycle was analyzed using an intercalating fluorochrome to assess cell DNA content. In analyses of yeast cultures, the presence of a sub-G1 peak in the fluorescent signal is often interpreted as a loss of DNA due to its fragmentation associated with apoptosis. However, the cell wall and its stucture may interfere with the fluorescent signal recorded. These observations indicate that misinterpretation of yeast DNA profiles is possible in analyses based on some of the most common probes: cells in G0 appeared to have a lower DNA content and may have been mistaken as a sub-G1 population. However, careful selection of the fluorochrome for DNA quantification allowed a direct discrimination between G0 and G1 yeast cell cycle steps, without additional labeling. We present and discuss results obtained with five current fluorochromes. These observations led us to recommend to use SYTOX Green for cycle analysis of living cells and SYBR Green I for the identification of the apoptosis sub-G1 population identification or the DNA ploidy application.

PMID:
24392149
PMCID:
PMC3879310
DOI:
10.1371/journal.pone.0084645
[Indexed for MEDLINE]
Free PMC Article

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