analysis of the gfp-positive and mcherry-positive cells by flow cytometry

对GFP和mCherry报告细胞进行FACS分析，以评估NHEJ和HR效率

A DNA double-strand break (DSB) is the most deleterious form of DNA damage, potentially leading to genome instability, chromosomal rearrangements, cellular senescence, and cell death if not repaired promptly1. Two well-established pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), are recognized for their effectiveness in addressing DNA DSBs2,3. HR is considered an error-free mechanism for DSB repair, utilizing homologous sequences in the sister chromatid as a template to restore the original configuration of the injured DNA molecule3. NHEJ, on the other hand, is an error-prone DSB repair pathway that joins the broken DNA ends without relying on any template2.
The NHEJ assay and HR assay are classical methods originally developed in Jasin&#39;s laboratory at Memorial Sloan-Kettering Cancer Center and utilized to quantify the efficiency of NHEJ and HR, respectively4,5,6,7. These assays play a crucial role in investigating the relevant mechanisms and factors associated with NHEJ and HR8,9,10,11,12,13,14. Both assays rely on the implementation of two disrupted GFP reporters, EJ5-GFP and DR-GFP, to monitor the repair of DSBs induced by the I-SceI endonuclease. The EJ5-GFP reporter is employed in the NHEJ assay, while the DR-GFP reporter is utilized in the HR assay. Each reporter is subtly designed so that the I-SceI-induced DSBs can only be repaired by a specific repair pathway to restore a GFP expression cassette4,5.
The NHEJ assay and HR assay can be conducted using either a chromosomally integrated or an extrachromosomal approach15,16. The chromosomally integrated approach necessitates the integration of the disrupted GFP reporters into the genome, allowing the analysis of DSB repair within a chromosomal context6,15. However, this approach requires prolonged cell passaging and is unsuitable for comparative studies involving multiple cell lines due to arbitrary chromosomal integration, introducing an additional confounding factor apart from inherent differences. In this protocol, we describe the extrachromosomal NHEJ assay and HR assays, involving the transient transfection of the disrupted GFP and I-SceI plasmids into HEK-293T cells, followed by flow cytometry analysis (the experiment workflow is shown in Figure 1). These non-integrated reporter assays were originally reported by Jasin&#39;s laboratory to study DNA interstrand cross-links repair16 and have been employed to assess NHEJ efficiency and HR efficiency by several laboratories9,10,11,12,13,14,17,18,19, including ours11. These extrachromosomal approaches facilitate the analysis of DSB repair in comparative studies involving multiple established stable cell lines.

作者聚焦：通过染色体外 NHEJ 检测和 HR 检测解码 DNA 修复

使用基于 GFP 的报告系统分析 HEK-293T 细胞中的非同源末端连接和同源重组效率

DNA double-strand breaks represent the most perilous DNA lesions. In response, cells employ two primary mechanisms for DNA double-strand break repair, NHEJ and HR.Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with them. The NHEJ assay and the HR assay are established methods used to measure the efficiency of NHEJ and HR.These methods rely on meticulously designed plasmids that contain a disrupted green fluorescent protein gene with recognition sites for endonuclease I-SceI for induction of DSBs.The NHEJ assay and the HR assay can be conducted using is a chromosomally-integrated or a extrachromosomal approach. The chromosomally-integrated approach enables the analysis of DSB repair within a chromosomally contest. However, the chromosomally integrated approach requires prolonged cell and is unsuitable for comparative studies involving multiple cell lines.In this protocol, we describe the extrachromosomal NHEJ and HR assays involving transient transfection of the disrupted GFP and I-SceI plasmids into HEK-293T cells and subsequent flow cytometry analysis. These nonintegrated reporter assays have been employed to assess the NHEJ efficiency and HR efficiency by several labs, including ours. In contrast to the chromosomally-integrated approach, this extrachromosomal approach enables swift analysis of the NHEJ or HR efficiency through the utilization of established, stable cell lines.

109 Views.  Qiqihar Medical University.  Analysis of the GFP-Positive and mCherry-Positive Cells by Flow Cytometry

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109 Views.  Qiqihar Medical University. Analysis of the GFP-Positive and mCherry-Positive Cells by Flow Cytometry

Video: Analysis of the GFP-Positive and mCherry-Positive Cells by Flow Cytometry

通过流式细胞术分析 GFP 阳性和 mCherry 阳性细胞

首先转染 HEK 293 T 细胞。转染后三天，从孔中取出培养基，并用 PBS 洗涤一次。向每个孔中加入 500 微升胰蛋白酶。在 37 摄氏度下孵育 5 分钟，以从板上分离所有细胞。然后向每个孔中加入 500 微升完全 DMEM 培养基并重悬细胞。将每个孔中的所有细胞转移到 1.5 毫升离心管中。然后在室温下以 1， 000 g 离心两分钟。通过移液小心地去除上清液，并用一毫升PBS洗涤沉淀一次。再次，小心地除去上清液并将细胞重悬于500微升PBS中。将细胞转移到FACS管中。使用流式细胞仪测定细胞。加载未切除的细胞悬液。在流式细胞术工作表上，按“获取”并调整正向和侧向散射电压。要将像元定位在中间区域，请在该群体 R1 周围设门，以排除左下象限的碎片。将 FL1、FL2 点印迹的闸门更改为 G R1。选择四门控工具，然后单击 FL1/FL2 点印迹中细胞群的右上极值作为 G R1。调整 FL1/FL2 电压，将电池放置在左下象限。按暂停和中止。然后加载 GFP 单色对照池。按 Acquire 并调整补偿，将 GFP 阳性细胞定位在右下象限。按暂停和中止。接下来，加载 mCherry 单色对照细胞。按 Acquire 并调整补偿，将 mCherry 阳性细胞置于左上象限。按暂停和中止。单击菜单栏上的 Acquire，然后选择 Acquisition Storage。在“收集条件”下，选择“当计算 G1 R1 事件的 10， 000 个时，采集将停止”。取消“设置”的复选标记。加载样品池。按 Acquire 并等待，直到计数完 10， 000 个 G1 R1 细胞。对其他样品和对照重复相同的操作。

analysis-gfp-positive-mcherry-positive-cells-flow

Research

JoVE Journal

Biology

Analysis of the GFP-Positive and mCherry-Positive Cells by Flow Cytometry

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.

This protocol describes an extrachromosomal nonhomologous end joining (NHEJ) assay and homologous recombination (HR) assay to quantify the efficiency of NHEJ and HR in HEK-293T cells.

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In ...