Flow cytometric determination of cell cycle progression via direct labeling of replicated DNA
Flow cytometric determination of cell cycle progression via direct labeling of replicated DNA
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DOI：
10.1016/j.ab.2020.114002
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发表时间：
2021-02-01
影响因子：
2.9
通讯作者：
Kraus, Tomas
中科院分区：
文献类型：
--
作者：
Kuzmova, Erika;Zawada, Zbigniew;Kraus, Tomas
报道的方法允许在体外真核细胞中的DNA复制和细胞周期进程的简单和快速的监测。通过掺入代谢活性荧光（Cy 3）脱氧尿苷三磷酸衍生物标记复制细胞的DNA，该衍生物通过合成转运蛋白（SNTT 1）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分析。因此，该方案避免了后标记步骤，这在目前使用的掺入测定（BrdU，EdU）中是必不可少的。在贴壁（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（包括受DNA聚合酶抑制剂（阿非迪霉素）影响的细胞）的细胞周期分析中证明了该方案的适用性。由于掺入时间短（5-60 min）和步骤数减少，该方案可在1-2 h内完成，细胞损失最小，重现性极佳。
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.