Protocol to image and quantify nucleocytoplasmic transport in cultured cells using fluorescent in situ hybridization and a dual reporter system.
Protocol to image and quantify nucleocytoplasmic transport in cultured cells using fluorescent in situ hybridization and a dual reporter system.
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DOI：
10.1016/j.xpro.2022.101813
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发表时间：
2022-12-16
期刊：
影响因子：
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通讯作者：
Ding, Baojin
中科院分区：
文献类型：
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作者：
Cui, Haochen;Sepehrimanesh, Masood;Coutee, Casey A;Akter, Masuma;Hosain, Md Abir;Ding, Baojin
核质转运（NCT）在维持细胞内稳态中起着重要作用。在这里，我们提出了一个协议来测量NCT的转录和蛋白质货物在培养的细胞。我们首先描述了荧光原位杂交（FISH）测定来测量核mRNA输出。然后，我们详细介绍了双报告系统来测量蛋白质NCT。该方案还包括使用CellProfilerTM的图像分析和数据输出。该组合方法可用于无偏地分析培养细胞中的NCT活性。有关本方案使用和执行的完整详细信息，请参阅。通过荧光原位杂交（FISH）测量核mRNA输出使用双报告系统检查蛋白质输入和输出活动使用开源软件量化图像检查和量化培养细胞（包括神经元）中的NCT活动出版商说明：进行任何实验方案都需要遵守当地机构的实验室安全和道德准则。核质转运（NCT）在维持细胞内稳态中起着重要作用。在这里，我们提出了一个协议来测量NCT的转录和蛋白质货物在培养的细胞。我们首先描述了荧光原位杂交（FISH）测定来测量核mRNA输出。然后，我们详细介绍了双报告系统来测量蛋白质NCT。该方案还包括使用CellProfilerTM的图像分析和数据输出。该组合方法可用于无偏差地分析培养细胞中的NCT活性。
Nucleocytoplasmic transport (NCT) plays critical roles in maintaining cellular homeostasis. Here, we present a protocol to measure NCT for both transcript and protein cargos in cultured cells. We first describe the fluorescent in situ hybridization (FISH) assay to measure the nuclear mRNA export. We then detail a dual reporter system to measure the protein NCT. This protocol also includes image analysis and data output using CellProfiler™. The combined approach can be used to unbiasedly analyze NCT activities in cultured cells. For complete details on the use and execution of this protocol, please refer to. Measure nuclear mRNA export by fluorescent in situ hybridization (FISH) Examine protein import and export activities with a dual reporter system Use an open-source software to quantify images Examine and quantify NCT activities in cultured cells, including neurons Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Nucleocytoplasmic transport (NCT) plays critical roles in maintaining cellular homeostasis. Here, we present a protocol to measure NCT for both transcript and protein cargos in cultured cells. We first describe the fluorescent in situ hybridization (FISH) assay to measure the nuclear mRNA export. We then detail a dual reporter system to measure the protein NCT. This protocol also includes image analysis and data output using CellProfiler™. The combined approach can be used to unbiasedly analyze NCT activities in cultured cells.