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

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.