Protocol to image and quantify nucleocytoplasmic transport in cultured cells using fluorescent in situ hybridization and a dual reporter system

Abstract

Summary: 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 Ding et al. (2020, 2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Cell culture
• Cell-based Assays
• Gene Expression
• In Situ Hybridization
• Microscopy