Background

Understanding the temporal regulation of cell cycle progression provides critical insight into cellular proliferation, differentiation, and the mechanisms driving pathological states, such as carcinogenesis [1–3]. Traditional flow cytometry has long been the standard for analyzing cell cycle distribution by quantifying DNA content [4,5]. While widely adopted, this method has key limitations. It relies on population-based measurements, necessitates large sample sizes, and poses technical challenges for achieving single-cell cell cycle stage identification and resolving sub-stages within the cell cycle.

Several earlier methods have aimed to improve upon conventional cell cycle analysis. One widely used approach is the FUCCI (fluorescent ubiquitination-based cell cycle indicator) system, which enables live-cell cell cycle identification by utilizing fluorescently tagged Cdt1 and Geminin [6–8]. The commonly used FUCCI system distinguishes G1 from S/G2/M phases. However, it requires transgenic expression and live-cell imaging, limiting its accessibility and broad applicability. Alternatively, EdU- or BrdU-based labeling assays with flow cytometry or microscopy provide improved detection of the S phase by identifying incorporated nucleotide analogs into replicating DNA [5]. However, these approaches require careful optimization of incorporation timing and duration and may introduce cytotoxic effects.

To overcome these limitations, the ImmunoCellCycle-ID method was developed (Figure 1) [1]. This method leverages immunofluorescence-based techniques using four endogenous markers, DNA, proliferating cell nuclear antigen (PCNA), centromere protein F (CENP-F), and centromere protein C (CENP-C), to enable high-resolution cell cycle identification at the single-cell level. Remarkably, it allows identification of G1, early S, late S, early G2, late G2, and sub-stages of M phase.

PCNA, a key factor in DNA replication, displays distinct nuclear localization patterns across the cell cycle [1,9]. In the G1 phase, PCNA is uniformly distributed within the nucleus. In the early S phase, it forms distinct small puncta, which then grow into larger aggregates during the late S phase, return to a uniform distribution in the G2 phase, and become markedly reduced during mitosis (Figure 2) [1]. CENP-F, a kinetochore-associated protein, localizes to kinetochores from prophase through anaphase [1,10–13]. Interestingly, it begins accumulating in the nucleus from the S phase and progressively increases in the G2 phase, despite the underlying mechanism remaining unclear [1,13]. Therefore, co-staining with PCNA and CENP-F enables reliable identification of the G1, early S, late S, and G2 phases without requiring quantification. Specifically, G1 nuclei are CENP-F negative with weak, uniform PCNA; G2 nuclei are CENP-F positive with similarly uniform PCNA (Figure 2). The presence of PCNA nuclear puncta (early/late S phase) always coincides with CENP-F positivity, eliminating contamination from the G1 phase. CENP-C localizes to centromeric chromatin throughout the cell cycle [1,14,15] and serves as a marker for distinguishing between early and late G2 phases (Figure 2). In the early G2 phase, kinetochores remain closely paired within diffraction-limited distances, whereas in the late G2 phase, they separate into distinct paired foci (Figure 2) [1]. This morphological change allows accurate distinction between these two stages without quantification. Mitotic sub-stages can be readily identified based on characteristic DNA morphologies (Figure 2).

Overall, ImmunoCellCycle-ID offers a high-precision, user-friendly, and cost-effective alternative to traditional cell cycle analysis methods. It is flexible and compatible with co-labeling of additional cellular markers, enabling dynamic studies of target proteins throughout the cell cycle. Furthermore, with alternative markers, this method can be extended to include G0 phase identification [1,16,17]. In this protocol, we focus on identifying the G1, early S, late S, early G2, late G2, and M phases in asynchronous RPE1 cells using the standard ImmunoCellCycle-ID method. The RPE1 cell line was selected due to its non-transformed, normal epithelial origin, stable diploid karyotype, and wild-type p53 status, making it a widely used control model in cell cycle research. In our original manuscript [1], we demonstrated that the ImmunoCellCycle-ID method is not only effective in RPE1 cells but also robustly applicable to a variety of cell lines, including HCT116 (colon cancer), CAL-51 (triple-negative breast cancer), T47D (luminal A breast cancer), HeLa (cervical cancer), and U2OS (osteosarcoma), without the need for modifications to the staining protocol. A representative example of standard ImmunoCellCycle-ID images in asynchronous CAL-51 cells is shown in Figure 3.

Figure 1. Cell cycle distribution in asynchronous RPE1 cells. Representative ImmunoCellCycle-ID images of asynchronous RPE1 cells, with corresponding identification of their cell cycle stages. All images were visualized using maximum z-projection.

Figure 2. Representative ImmunoCellCycle-ID images for each cell cycle stage. Representative immunofluorescence images showing labeling of CENP-C, PCNA, CENP-F, and DAPI across all stages of the cell cycle. All images were adjusted to the same contrast settings across all cell cycle stages and visualized using maximum z-projection.


Figure 3. Cell cycle distribution in asynchronous CAL-51 cells. Representative ImmunoCellCycle-ID images of asynchronous CAL-51 cells, with corresponding identification of their cell cycle stages. All images were visualized using maximum z-projection.