A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-G1
A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-G1
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DOI：
10.1371/journal.pone.0084645
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发表时间：
2014-01-02
期刊：
影响因子：
3.7
通讯作者：
Tesniere, Catherine
中科院分区：
文献类型：
--
作者：
Delobel, Pierre;Tesniere, Catherine
流式细胞术广泛用于医学和临床应用，现在正用于食品微生物学的各种应用。流式细胞术在酵母细胞中的大多数应用都来自于哺乳动物细胞的方法，但酵母细胞可能存在特异性，必须考虑对数据输出进行严格分析，以避免任何误解。我们报告了酿酒酵母细胞周期进程的分析，突出了可能的错误。使用嵌入荧光染料分析细胞周期以评估细胞DNA含量。在酵母培养物的分析中，荧光信号中亚G1峰的存在通常被解释为DNA的损失，这是由于其与细胞凋亡相关的片段化。然而，细胞壁及其结构可能会干扰所记录的荧光信号。这些观察结果表明，在基于一些最常见的探针的分析中，可能会误解酵母DNA谱：G0中的细胞似乎具有较低的DNA含量，可能被误认为是亚G1群体。然而，仔细选择的DNA定量荧光染料允许G0和G1酵母细胞周期步骤之间的直接歧视，没有额外的标记。我们提出并讨论了五个电流荧光染料得到的结果。这些观察结果使我们推荐使用SYTOX绿色用于活细胞的周期分析，SYBR绿色I用于鉴定凋亡亚G1群体鉴定或DNA倍性应用。
Extensively developed for medical and clinical applications, flow cytometry is now being used for diverse applications in food microbiology. Most uses of flow cytometry for yeast cells are derived from methods for mammalian cells, but yeast cells can present specificities that must be taken into account for rigorous analysis of the data output to avoid any misinterpretation. We report an analysis of Saccharomyces cerevisiae cell cycle progression that highlights possible errors. The cell cycle was analyzed using an intercalating fluorochrome to assess cell DNA content. In analyses of yeast cultures, the presence of a sub-G1 peak in the fluorescent signal is often interpreted as a loss of DNA due to its fragmentation associated with apoptosis. However, the cell wall and its stucture may interfere with the fluorescent signal recorded. These observations indicate that misinterpretation of yeast DNA profiles is possible in analyses based on some of the most common probes: cells in G0 appeared to have a lower DNA content and may have been mistaken as a sub-G1 population. However, careful selection of the fluorochrome for DNA quantification allowed a direct discrimination between G0 and G1 yeast cell cycle steps, without additional labeling. We present and discuss results obtained with five current fluorochromes. These observations led us to recommend to use SYTOX Green for cycle analysis of living cells and SYBR Green I for the identification of the apoptosis sub-G1 population identification or the DNA ploidy application.