Nuclear Histone H3 Post-translational Modification Profiling in Whole Cells using Spectral Flow Cytometry

Discerning the dynamic nature of nuclear histone H3 post-translational modifications (H3-PTMs) is crucial for understanding gene regulatory mechanisms. Although conventional nuclear H3-PTM flow cytometry protocols currently offer acceptable sensitivity, they typically involve isolating nuclei prior to running the assay, leading to significant sample loss and limited throughput. To address these challenges, we present a high-throughput nuclear H3-PTM spectral flow cytometry protocol conducted directly in whole cells. By performing fixation, permeabilization, and antibody incubations in a 96-well plate format, we eliminate the need for nuclei isolation, thereby preserving sample integrity and increasing throughput. Notably, this protocol allows for the simultaneous assessment of histone PTMs, cytoplasmic proteins, plasma membrane receptors, and cell cycle analysis within the same cell, providing comprehensive insights into the status and functional utility of a cell. This approach not only streamlines experimental procedures but also enhances the efficiency and reproducibility of nuclear flow cytometry assays, allowing for a deeper exploration of epigenetic regulation across diverse biological samples.