All proliferative eukaryotic cells navigate the cell cycle - a complex, highly regulated process that progresses through four distinct phases: G1 (first growth or gap phase), S (DNA replication), G2 (second growth or gap phase) and M (mitosis). Additionally, cells can transition to a reversible quiescent state known as G0. Here (Chen et al., 2024), Aussie Suzuki and colleagues develop an immunofluorescence-based method (ImmunoCellCycle-ID) that accurately identifies cell cycle phases in fixed cells with high precision and single-cell resolution. The authors evaluate several cell cycle proteins in the hTert-RPE1 cell line and find that staining for the specific combination of CENP-C, CENP-F and PCNA enables precise discrimination across all cell cycle stages; DNA staining differentiates cells in M phase, and PCNA and CENP-F effectively distinguish between G1, S and G2 phases, with CENP-C providing further specificity for early and late G2 substages. In addition, ImmunoCellCycle-ID exhibits a robust phase detection rate that is comparable to that of flow cytometry, while achieving finer resolution of S and G2 substages. The authors validate their method across several transformed cell lines, revealing cell cycle distribution patterns consistent with those found by flow cytometry. Overall, this new method offers a powerful tool for studying cell cycle dynamics, particularly in cancer research, enhancing our understanding of cellular proliferation and regulation.
