Patterson James O, Swaffer Matthew, Filby Andrew
Cell Cycle Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, Holborn WC2A 3LY, UK.
FACS Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, Holborn WC2A 3LY, UK; Flow Cytometry Core Facility, Newcastle Biomedicine, Newcastle University, Newcastle-upon-Tyne NE1 7RU, UK.
Methods. 2015 Jul 1;82:74-84. doi: 10.1016/j.ymeth.2015.04.026. Epub 2015 May 4.
Fission yeast (Schizosaccharomyces pombe) is an excellent model organism for studying eukaryotic cell division because many of the underlying principles and key regulators of cell cycle biology are conserved from yeast to humans. As such it can be employed as tool for understanding complex human diseases that arise from dis-regulation in cell cycle controls, including cancers. Conventional Flow Cytometry (CFC) is a high-throughput, multi-parameter, fluorescence-based single cell analysis technology. It is widely used for studying the mammalian cell cycle both in the context of the normal and disease states by measuring changes in DNA content during the transition through G1, S and G2/M using fluorescent DNA-binding dyes. Unfortunately analysis of the fission yeast cell cycle by CFC is not straightforward because, unlike mammalian cells, cytokinesis occurs after S-phase meaning that bi-nucleated G1 cells have the same DNA content as mono-nucleated G2 cells and cannot be distinguished using total integrated fluorescence (pulse area). It has been elegantly shown that the width of the DNA pulse can be used to distinguish G2 cells with a single 2C foci versus G1 cells with two 1C foci, however the accuracy of this measurement is dependent on the orientation of the cell as it traverses the laser beam. To this end we sought to improve the accuracy of the fission yeast cell cycle analysis and have developed an Imaging Flow Cytometry (IFC)-based method that is able to preserve the high throughput, objective analysis afforded by CFC in combination with the spatial and morphometric information provide by microscopy. We have been able to derive an analysis framework for subdividing the yeast cell cycle that is based on intensiometric and morphometric measurements and is thus robust against orientation-based miss-classification. In addition we can employ image-based metrics to define populations of septated/bi-nucleated cells and measure cellular dimensions. To our knowledge, this is the first use of IFC to study fission yeast and we are confident that this will provide a springboard for further IFC-based analysis across all aspects of fission yeast biology.
裂殖酵母（粟酒裂殖酵母）是研究真核细胞分裂的优秀模式生物，因为细胞周期生物学的许多基本原理和关键调节因子在从酵母到人类的过程中都是保守的。因此，它可以作为一种工具，用于理解由细胞周期控制失调引起的复杂人类疾病，包括癌症。传统流式细胞术（CFC）是一种基于荧光的高通量、多参数单细胞分析技术。它通过使用荧光DNA结合染料测量在G1、S和G2/M转换过程中DNA含量的变化，广泛用于研究正常和疾病状态下的哺乳动物细胞周期。不幸的是，用CFC分析裂殖酵母细胞周期并非易事，因为与哺乳动物细胞不同，胞质分裂发生在S期之后，这意味着双核G1细胞与单核G2细胞具有相同的DNA含量，无法使用总积分荧光（脉冲面积）进行区分。已经巧妙地证明，DNA脉冲的宽度可用于区分具有单个2C焦点的G2细胞与具有两个1C焦点的G1细胞，然而这种测量的准确性取决于细胞穿过激光束时的方向。为此，我们试图提高裂殖酵母细胞周期分析的准确性，并开发了一种基于成像流式细胞术（IFC）的方法，该方法能够保持CFC提供的高通量、客观分析，并结合显微镜提供的空间和形态测量信息。我们已经能够推导出一个基于强度测量和形态测量的酵母细胞周期细分分析框架，因此对基于方向的错误分类具有鲁棒性。此外，我们可以使用基于图像的指标来定义分隔/双核细胞群体并测量细胞尺寸。据我们所知，这是首次使用IFC研究裂殖酵母，我们相信这将为基于IFC的裂殖酵母生物学各方面的进一步分析提供一个跳板。
