Work

Protocol for quantifying phase-separated condensates in living cells using HILO microscopy imaging and genetic engineering

Year: 2025

Type: article

Abstract: Spatial clustering of epigenetic factors through phase separation is an emerging concept in transcriptional regulation and genome organization. Here, we present a protocol to quantify the phase separation capacity of epigenetic factors in living cells via HILO (highly inclined and laminated optical sheet) microscopy. We describe steps for using CRISPR-Cas9 to generate a cell line with two distinct fusion proteins containing fluorophores and either CRE-LoxP- or dTAG-inducible degradation systems. We then detail the procedures for imaging fusion proteins to analyze and quantify phase separation capacity. For complete details on the use and execution of this protocol, please refer to Brown et al. 1 • Steps for creating cell lines with two distinct fluorescent fusion proteins • Procedures utilizing Cre-LoxP or dTag degradation systems • Guidance for single-molecule HILO microscopy and CellProfiler analysis • Instructions for measuring condensed fraction of phase-separated proteins Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Spatial clustering of epigenetic factors through phase separation is an emerging concept in transcriptional regulation and genome organization. Here, we present a protocol to quantify the phase separation capacity of epigenetic factors in living cells via HILO microscopy. We describe steps for using CRISPR-Cas9 to generate a cell line with two distinct fusion proteins containing fluorophores and either CRE-LoxP- or dTAG-inducible degradation systems. We then detail the procedures for imaging fusion proteins to analyze and quantify phase separation capacity.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104151

Authors: Axel Espinoza, Steven Ingersoll, Xiaojun Ren

Institutions: University of Colorado Denver, University of Colorado Anschutz, University of North Carolina at Charlotte

Language: English

Topic: Genomics and Chromatin Dynamics

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,550

Funder: National Institute of General Medical Sciences