Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry

Abstract

Summary: Chromatin-bound proteins have been conventionally measured through subcellular fractionation followed by immunoblotting or by immunofluorescence microscopy. Here, we present Chromoflow, a protocol for the quantitative analyses of protein levels on chromatin in single cells and throughout the cell cycle using flow cytometry. We describe steps for harvesting cells and for nuclear extraction, and a barcoding strategy to multiplex samples from different conditions that reduces antibody staining variability and eliminates the need for normalization.1,2 We then detail procedures for data acquisition and analysis.For complete details on the use and execution of this protocol, please refer to Alonso-Gil et al. (2023).3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Flow Cytometry/Mass Cytometry
• Molecular Biology