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

• Published in: STAR protocols 2023-12, Vol.4 (4), p.102568-102568, Article 102568
• Main Authors: Alonso-Gil, Dácil, Losada, Ana
• Format: Article
• Language: English
• Subjects: Antibodies; Cell Biology; Cell Cycle; Cell Division; Chromatin - genetics; Flow Cytometry; Flow Cytometry/Mass Cytometry; Molecular Biology; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102568

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 [Display omitted] •Fast and reproducible analysis of chromatin-bound proteins throughout the cell cycle•Sample multiplexing avoids antibody staining variability and facilitates comparisons•Flow cytometry data obtained by Chromoflow are highly versatile and easy to quantify Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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. We then detail procedures for data acquisition and analysis.