Abstract
The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G1 and G2 phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G1 phase. We have devised a flow cytometric method exploiting the fact that cells in G1 phase contain two nuclei, whereas cells in G2 are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G1 and in G2 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.
Highlights
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]
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)
The major problem in analyzing Schzosaccharomyces pombe cells and their cell cycle by flow cytometry is that they contain the same amount of DNA in G1 and in G2 phase
Summary
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. The major problem in analyzing Schzosaccharomyces pombe cells and their cell cycle by flow cytometry is that they contain the same amount of DNA in G1 and in G2 phase.
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