A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-G1
Tesnie`re C (2014) A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-
G1. PLoS ONE 9(1): e84645. doi:10.1371/journal.pone.0084645
A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-G1
Pierre Delobel 0
Catherine Tesnie` re 0
Michael Polymenis, Texas A&M University, United States of America
0 1 INRA, UMR1083, Montpellier, France, 2 SupAgro Montpellier, UMR1083, Montpellier, France , 3 Universit Montpellier 1, UMR1083, Montpellier , France
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.
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The yeast Saccharomyces cerevisiae has been used as a model system
for the study of the eukaryotic cell cycle [1]. Its main experimental
advantage is the ease with which the cell cycle can be analyzed by
simple culture techniques. In a proliferating population, the
distribution of cells within the major distinct phases of the cell
cycle (Figure 1) can be determined from differences in DNA
content between cells in prereplicative phases (G0 and G1), cells
replicating DNA (S), and post-replicative plus mitotic (G2+M)
phase cells. Cells in G2 and M phases have identical DNA content
and thus cannot be discriminated on this basis. Cells with
fractional DNA content, a situation occuring during apoptosis,
can be identified as a sub-G1 population [2]. The progression of
the cycle in yeast cells can be investigated by flow cytometry
(FCMFCMFlow cytometry) following fixation and staining with a
fluorescent dye that binds stoichiometrically to DNA. FCM is a
powerful and widely used approach to estimating cell cycle phase
distribution and analyzing many aspects of cell cycle regulation in
diverse cell types [3]. However, some of the specificities of yeast
need be taken into account when using this technique. For
instance, the percentage of each yeast population represents the
time spent in each phase: in a rich medium during exponential
growth G1 phase represents only 1=4 of the total cycle, whereas
the S-G2/M phases last about 3=4 of the cell cycle [4]. In addition,
doublets and gating difficulties can be encountered in this budding
yeast population when analyzed by FCM.
Fluorochromes have different characteristics that can influence
the evaluation of DNA content and help to improve assessments of
the yeast cell cycle. For instance, some fluorochromes are DNA
specific or not, GC selective or general intercalating dyes. Thus,
we tested and compared five current fluorochromes (SYTOX
Green, propidium iodide [PI]PIpropidium iodide, TO-PRO-3,
7aminoactinomycin D [7-AAD]7-AAD7-aminoactinomycin D and
SYBR Green I) that could resolve DNA quantification and/or cell
cycle status. For that, a single cell preparation was performed,
including RNase treatment. Then, the preparation was labeled by
each fluorochrome individually.
We report a critical analysis of evaluations of the budding yeast
cell cycle by FCM with single DNA staining, and their
interpretation. We indicate how the problems identified can be
solved.
Growth Kinetics
Cytometry measurements giving aberrant data concerning the
presence of sub-G1 populations in S. cerevisiae samples (data not
shown) led us to analyze the method for studying this yeast cell
cycle. In particular, we looked at the choice of the fluorochrome
using standard cultures.
To identify the different cell growth stages, we analyzed culture
growth kinetics by following cell density during a single culture in
standard Y (...truncated)