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Cell-cycle analysis of fission yeast cells by flow cytometry.

Knutsen JH, Rein ID, Rothe C, Stokke T, Grallert B, Boye E - PLoS ONE (2011)

Bottom Line: This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase.Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase.Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.

ABSTRACT
The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.

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Schematic representation of the S. pombe cell cycle.The circle indicates the relative positions and durations of the different cell-cycle phases. The bodies outside the circle indicate the morphology of cells at the different phases and the numbers in parentheses show the subpopulations that they belong to (see Figure 2). The nuclei are indicated by dark spots.
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pone-0017175-g001: Schematic representation of the S. pombe cell cycle.The circle indicates the relative positions and durations of the different cell-cycle phases. The bodies outside the circle indicate the morphology of cells at the different phases and the numbers in parentheses show the subpopulations that they belong to (see Figure 2). The nuclei are indicated by dark spots.

Mentions: The fission yeast, Schizosaccharomyces pombe, is a popular model system, amenable to classic and molecular genetic analysis as well as biochemical and physiological studies [1]. The cell-cycle progression of fission yeast can be measured by flow cytometry, which is a powerful method to analyse many aspects of cell-cycle regulation for most organisms. However, because of two special features analysis of fission yeast cell growth by flow cytometry is not straightforward: First, under standard laboratory conditions the cytokinesis of fission yeast cells occurs at the end of S phase and for that reason cells in G1 and S phase are binuclear (Fig. 1). Cells in G1 phase contain two nuclei, each with a single, complete genome (termed 1C DNA) and these cells contain the same total amount of DNA (2C) as cells in G2 phase, which harbor their DNA in a single nucleus. Therefore, the discrimination of G1 cells from G2 cells is not straightforward by simple measurements of the cellular DNA content in a flow cytometer. Second, the fission yeast cells tend to form multimers by sticking together, thereby perturbing flow cytometric analyses of single-cell behaviour and of cell-cycle kinetics. Here we show how these problems can be solved. The methods presented are technically simple and available to most flow cytometry users. We also give examples of useful applications of the novel methods.


Cell-cycle analysis of fission yeast cells by flow cytometry.

Knutsen JH, Rein ID, Rothe C, Stokke T, Grallert B, Boye E - PLoS ONE (2011)

Schematic representation of the S. pombe cell cycle.The circle indicates the relative positions and durations of the different cell-cycle phases. The bodies outside the circle indicate the morphology of cells at the different phases and the numbers in parentheses show the subpopulations that they belong to (see Figure 2). The nuclei are indicated by dark spots.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046126&req=5

pone-0017175-g001: Schematic representation of the S. pombe cell cycle.The circle indicates the relative positions and durations of the different cell-cycle phases. The bodies outside the circle indicate the morphology of cells at the different phases and the numbers in parentheses show the subpopulations that they belong to (see Figure 2). The nuclei are indicated by dark spots.
Mentions: The fission yeast, Schizosaccharomyces pombe, is a popular model system, amenable to classic and molecular genetic analysis as well as biochemical and physiological studies [1]. The cell-cycle progression of fission yeast can be measured by flow cytometry, which is a powerful method to analyse many aspects of cell-cycle regulation for most organisms. However, because of two special features analysis of fission yeast cell growth by flow cytometry is not straightforward: First, under standard laboratory conditions the cytokinesis of fission yeast cells occurs at the end of S phase and for that reason cells in G1 and S phase are binuclear (Fig. 1). Cells in G1 phase contain two nuclei, each with a single, complete genome (termed 1C DNA) and these cells contain the same total amount of DNA (2C) as cells in G2 phase, which harbor their DNA in a single nucleus. Therefore, the discrimination of G1 cells from G2 cells is not straightforward by simple measurements of the cellular DNA content in a flow cytometer. Second, the fission yeast cells tend to form multimers by sticking together, thereby perturbing flow cytometric analyses of single-cell behaviour and of cell-cycle kinetics. Here we show how these problems can be solved. The methods presented are technically simple and available to most flow cytometry users. We also give examples of useful applications of the novel methods.

Bottom Line: This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase.Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase.Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.

ABSTRACT
The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.

Show MeSH
Related in: MedlinePlus