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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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Related in: MedlinePlus

Monitoring cell-cycle progression through mitosis.Cell-cycle kinetics of a culture arrested in G2 phase and released into the cell cycle for 60 min. Cell doublets were eliminated by employing the gate shown in panel A, resulting in the DNA cytogram shown in panel B. Indicated are the positions of the binuclear cells and of cells in S phase. Panel C: quantification of the MI, BI, SI, the frequency of S-phase cells and of cells with chromatin-bound Mcms as a function of time after release. Results are shown for one representative experiment where all parameters were measured on the very same cell sample.
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pone-0017175-g005: Monitoring cell-cycle progression through mitosis.Cell-cycle kinetics of a culture arrested in G2 phase and released into the cell cycle for 60 min. Cell doublets were eliminated by employing the gate shown in panel A, resulting in the DNA cytogram shown in panel B. Indicated are the positions of the binuclear cells and of cells in S phase. Panel C: quantification of the MI, BI, SI, the frequency of S-phase cells and of cells with chromatin-bound Mcms as a function of time after release. Results are shown for one representative experiment where all parameters were measured on the very same cell sample.

Mentions: The ability to separate mononuclear from binuclear cells was exploited to monitor, by flow cytometry, the passage of fission yeast cells through mitosis. The standard method to achieve this involves microscopic measurements of the frequency of septating cells, the septation index (SI), and/or the frequency of cells that have passed into or through mitosis, the mitotic index (MI). Here we have employed flow cytometry to measure the frequency of binuclear cells (the binuclear index, BI) and compared the BI, the MI and the SI for one and the same cell culture synchronized in G2 phase and released into the cell cycle. After applying the gate to eliminate cell doublets (Figure 5A) we identified binuclear cells (Figure 5B), i.e. the cells found in subpopulations 2 and 3 of Figure 2A. The time and kinetics of rise in BI corresponded with the rise in MI and SI, but occurred in-between the MI and the SI (Figure 5C). We conclude that flow cytometric measurement of the BI is a convenient and simple method to estimate the time of mitotic entry.


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)

Monitoring cell-cycle progression through mitosis.Cell-cycle kinetics of a culture arrested in G2 phase and released into the cell cycle for 60 min. Cell doublets were eliminated by employing the gate shown in panel A, resulting in the DNA cytogram shown in panel B. Indicated are the positions of the binuclear cells and of cells in S phase. Panel C: quantification of the MI, BI, SI, the frequency of S-phase cells and of cells with chromatin-bound Mcms as a function of time after release. Results are shown for one representative experiment where all parameters were measured on the very same cell sample.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017175-g005: Monitoring cell-cycle progression through mitosis.Cell-cycle kinetics of a culture arrested in G2 phase and released into the cell cycle for 60 min. Cell doublets were eliminated by employing the gate shown in panel A, resulting in the DNA cytogram shown in panel B. Indicated are the positions of the binuclear cells and of cells in S phase. Panel C: quantification of the MI, BI, SI, the frequency of S-phase cells and of cells with chromatin-bound Mcms as a function of time after release. Results are shown for one representative experiment where all parameters were measured on the very same cell sample.
Mentions: The ability to separate mononuclear from binuclear cells was exploited to monitor, by flow cytometry, the passage of fission yeast cells through mitosis. The standard method to achieve this involves microscopic measurements of the frequency of septating cells, the septation index (SI), and/or the frequency of cells that have passed into or through mitosis, the mitotic index (MI). Here we have employed flow cytometry to measure the frequency of binuclear cells (the binuclear index, BI) and compared the BI, the MI and the SI for one and the same cell culture synchronized in G2 phase and released into the cell cycle. After applying the gate to eliminate cell doublets (Figure 5A) we identified binuclear cells (Figure 5B), i.e. the cells found in subpopulations 2 and 3 of Figure 2A. The time and kinetics of rise in BI corresponded with the rise in MI and SI, but occurred in-between the MI and the SI (Figure 5C). We conclude that flow cytometric measurement of the BI is a convenient and simple method to estimate the time of mitotic entry.

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