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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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The importance of light-scatter gating.Flow cytometric analyses of a culture of exponentially growing S. pombe cells (top half) and cells arrested in G1 phase (bottom). The figure displays two-parametric light scatter cytograms (A, F), two-parametric DNA cytograms (B, D, G, I) and DNA histograms (C, E, H, J). Shown are the entire populations (A, B, C and F, G, H) and the remaining cells when gating has been introduced (D, E and I, J), thus removing cell doublets. The gates are shown in panels A and F.
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pone-0017175-g004: The importance of light-scatter gating.Flow cytometric analyses of a culture of exponentially growing S. pombe cells (top half) and cells arrested in G1 phase (bottom). The figure displays two-parametric light scatter cytograms (A, F), two-parametric DNA cytograms (B, D, G, I) and DNA histograms (C, E, H, J). Shown are the entire populations (A, B, C and F, G, H) and the remaining cells when gating has been introduced (D, E and I, J), thus removing cell doublets. The gates are shown in panels A and F.

Mentions: The potential and power of the method to exclude cell doublets and to separate mononuclear from binuclear cells is illustrated in Figure 4, where we have analyzed an asynchronous (panels A–E) and a G1-synchronized (panels F–J) population of cells without (panels B, C, G, H) and with gating (panels D, E, I, J) on FSC and SSC. The reduction in the numbers of cells with 4C DNA for the asynchronous population (panel C versus E) and with 2C DNA for the synchronized population (panel H versus J) clearly shows that gating on this region in the FSC vs. SSC cytogram is necessary to obtain reliable DNA histograms of single cells and therefore to arrive at the correct cell-cycle distribution. Whereas the presence of cells with 4C DNA content in the ungated histogram (panel C) could suggest the presence of cells in G2 phase that had not yet passed through cytokinesis, gating on light scattering revealed that these cells were in fact cell doublets (panel E). Therefore, cytokinesis occurred before or at the S/G2 transition rather than in G2 phase. Sonication of the sample reduced the number of cell doublets, but not sufficiently to replace gating on FSC/SSC (Supplementary Figure S1). The true DNA content distributions of mononuclear and binuclear cells were obtained after additional gating with low and high DNA-W, respectively, as described above. After renormalization according to the percentage of cells with low and high DNA-W, the fractions of cells in the different compartments were obtained (Table 1). From these fractions, and the cell cycle time, it is possible to calculate the length of the G2, G1 and S phases. Table 1 shows that the population of cells with 2C DNA content in the G1- arrested culture contain cells in G1 phase that have not divided, but also a small fraction of cells in G2 phase.


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)

The importance of light-scatter gating.Flow cytometric analyses of a culture of exponentially growing S. pombe cells (top half) and cells arrested in G1 phase (bottom). The figure displays two-parametric light scatter cytograms (A, F), two-parametric DNA cytograms (B, D, G, I) and DNA histograms (C, E, H, J). Shown are the entire populations (A, B, C and F, G, H) and the remaining cells when gating has been introduced (D, E and I, J), thus removing cell doublets. The gates are shown in panels A and F.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017175-g004: The importance of light-scatter gating.Flow cytometric analyses of a culture of exponentially growing S. pombe cells (top half) and cells arrested in G1 phase (bottom). The figure displays two-parametric light scatter cytograms (A, F), two-parametric DNA cytograms (B, D, G, I) and DNA histograms (C, E, H, J). Shown are the entire populations (A, B, C and F, G, H) and the remaining cells when gating has been introduced (D, E and I, J), thus removing cell doublets. The gates are shown in panels A and F.
Mentions: The potential and power of the method to exclude cell doublets and to separate mononuclear from binuclear cells is illustrated in Figure 4, where we have analyzed an asynchronous (panels A–E) and a G1-synchronized (panels F–J) population of cells without (panels B, C, G, H) and with gating (panels D, E, I, J) on FSC and SSC. The reduction in the numbers of cells with 4C DNA for the asynchronous population (panel C versus E) and with 2C DNA for the synchronized population (panel H versus J) clearly shows that gating on this region in the FSC vs. SSC cytogram is necessary to obtain reliable DNA histograms of single cells and therefore to arrive at the correct cell-cycle distribution. Whereas the presence of cells with 4C DNA content in the ungated histogram (panel C) could suggest the presence of cells in G2 phase that had not yet passed through cytokinesis, gating on light scattering revealed that these cells were in fact cell doublets (panel E). Therefore, cytokinesis occurred before or at the S/G2 transition rather than in G2 phase. Sonication of the sample reduced the number of cell doublets, but not sufficiently to replace gating on FSC/SSC (Supplementary Figure S1). The true DNA content distributions of mononuclear and binuclear cells were obtained after additional gating with low and high DNA-W, respectively, as described above. After renormalization according to the percentage of cells with low and high DNA-W, the fractions of cells in the different compartments were obtained (Table 1). From these fractions, and the cell cycle time, it is possible to calculate the length of the G2, G1 and S phases. Table 1 shows that the population of cells with 2C DNA content in the G1- arrested culture contain cells in G1 phase that have not divided, but also a small fraction of cells in G2 phase.

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