Nuclear Histone 3 Post-Translational Modification Profiling in Whole Cells using Spectral Flow Cytometry.

Area of Science:

• Molecular Biology
• Cell Biology
• Epigenetics

Background:

• Histone modifications regulate gene expression and cell identity.
• Studying diverse histone post-translational modifications (PTMs) in isolation limits understanding of their coordinated roles.
• Conventional flow cytometry for histone PTMs has low multiplexing capacity and requires nuclear isolation, causing sample loss.

Purpose of the Study:

• To develop a novel spectral flow cytometry protocol, EpiFlow, for multiparametric analysis of histone PTMs in whole cells.
• To improve throughput, efficiency, and sample integrity compared to conventional methods.
• To provide an accessible and reproducible method for studying chromatin dynamics.

Main Methods:

• Developed EpiFlow, a spectral flow cytometry protocol for whole-cell histone PTM analysis.
• Utilized a 96-well plate format to enhance throughput.
• Preserved cellular integrity throughout the analysis process.

Main Results:

• EpiFlow successfully resolved subtle variations in histone PTMs within neural progenitor cells.
• The method captured distinct chromatin states across the cell cycle.
• Results were correlated with several cell markers, demonstrating comprehensive cellular state analysis.

Conclusions:

• EpiFlow offers a robust framework for exploring chromatin dynamics and histone PTMs.
• The protocol enhances understanding of cell identity and function through multiparametric analysis.
• Open-access resources ensure reproducibility and broad applicability in fundamental and therapeutic research.