Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo Nam. 2, CZ-16610, Prague 6, Czech Republic.
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo Nam. 2, CZ-16610, Prague 6, Czech Republic; University of Cambridge, Department of Chemistry, Lensfield Road, Cambridge, CB2 1EW, UK.
Anal Biochem. 2021 Feb 1;614:114002. doi: 10.1016/j.ab.2020.114002. Epub 2020 Nov 5.
The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5-60 min) and reduced number of steps, the protocol can be completed within 1-2 h with a minimal cell loss and with excellent reproducibility.
该报道的方法允许在体外对真核细胞中的 DNA 复制和细胞周期进程进行简单而快速的监测。通过将代谢活性荧光（Cy3）脱氧尿苷三磷酸衍生物掺入到细胞中来标记正在复制的细胞，该衍生物通过合成转运蛋白（SNTT1）递送到细胞中。然后将细胞固定，用 DAPI 染色并通过流式细胞术进行分析。因此，该方案避免了当前使用的掺入测定法（BrdU，EdU）中必不可少的标记后步骤。该方案在分析贴壁（U-2 OS，HeLa S3，RAW 264.7，J774 A.1，Chem-1，U-87 MG）和悬浮（CCRF-CEM，MOLT-4，THP-1，HL-60，JURKAT）细胞培养物的细胞周期时具有适用性，包括受 DNA 聚合酶抑制剂（aphidicolin）影响的细胞。由于掺入时间短（5-60 分钟）且步骤减少，因此该方案可以在 1-2 小时内完成，细胞损失最小，重复性好。
