Marina Bejarano Franco, Safia Boujataoui, Majd Hadji, Louis Hammer, Helle D Ulrich, L Maximilian Reuter
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引用次数: 0
Abstract
Flow cytometry is a versatile tool used for cell sorting, DNA content imaging, and determining various cellular characteristics. With the possibility of high-throughput analyses, it combines convenient labelling techniques to serve rapid, quantitative, and qualitative workflows. The ease of sample preparation and the broad range of applications render flow cytometry a preferred approach for many scientific questions. Yet, we lack practical adaptations to fully harness the quantitative and high-throughput capabilities of most cytometers for many organisms. Here, we present simple and advanced protocols for the analysis of total DNA content, de novo DNA synthesis, and protein association to chromatin in budding yeast and human cells. Upon optimization of experimental conditions and choice of fluorescent dyes, up to four parameters can be measured simultaneously and quantitatively for each cell of a population in a multi-well plate format. Reducing sample numbers, plastic waste, costs per well, and hands-on time without compromising signal quality or single-cell accuracy are the main advantages of the presented protocols. In proof-of-principle experiments, we show that DNA content increase in S-phase correlates with de novo DNA synthesis and can be predicted by the presence of the replicative helicase MCM2-7 on genomic DNA.
流式细胞仪是一种多功能工具,可用于细胞分拣、DNA 含量成像和确定各种细胞特征。流式细胞仪可以进行高通量分析,它结合了便捷的标记技术,可用于快速、定量和定性的工作流程。样本制备的简便性和广泛的应用范围使流式细胞仪成为许多科学问题的首选方法。然而,我们缺乏实用的适应性,无法充分利用大多数流式细胞仪的定量和高通量功能来研究许多生物。在此,我们将介绍用于分析萌芽酵母和人类细胞中总 DNA 含量、DNA 新合成以及蛋白质与染色质关联的简单和高级方案。通过优化实验条件和荧光染料的选择,可以在多孔板格式下同时定量测量群体中每个细胞的多达四个参数。在不影响信号质量或单细胞准确性的前提下,减少样品数量、塑料废弃物、每孔成本和操作时间是本方案的主要优势。在原理验证实验中,我们发现 S 期 DNA 含量的增加与新 DNA 合成有关,并可通过基因组 DNA 上复制螺旋酶 MCM2-7 的存在进行预测。
期刊介绍:
Biological Chemistry keeps you up-to-date with all new developments in the molecular life sciences. In addition to original research reports, authoritative reviews written by leading researchers in the field keep you informed about the latest advances in the molecular life sciences. Rapid, yet rigorous reviewing ensures fast access to recent research results of exceptional significance in the biological sciences. Papers are published in a "Just Accepted" format within approx.72 hours of acceptance.