Cell Cycle Analysis in HeLa Cells

This application note demonstrates how Cytely classifies cell cycle stages using morphological features derived from a standard nuclear stain in adherent HeLa cells. The approach combines automated segmentation with interactive gating to isolate a pure mitotic population while preserving spatial context.

Key features demonstrated in this note:

  • Visualizing morphological heterogeneity across a cell population
  • Identifying cell cycle phases (G0/G1, S/G2, M) using nuclear morphology
  • Sequential gating to isolate and quantify mitotic cells with high purity
  • Complex cell cycle analysis without specialized dyes; supports retrospective analysis

Experimental Method

HeLa cells were cultured and stained with a standard nuclear dye. Imaging was performed on adherent cells to preserve morphology. Cytely processed images using automated object detection (nuclei) and measurement of morphological parameters (e.g., area, circularity, major/minor axes, intensity).

Complete experimental and analysis workflow
Figure 1: Complete experimental and analysis workflow. The process begins with cell culture and fluorescent labeling, followed by image acquisition. Cytely then performs automated segmentation and feature extraction, enabling interactive gating and analysis to deliver final population quantification.

Data Analysis in Cytely

  1. Upload: Images were added via Cytely’s web interface.
  2. Segmentation: Automated detection of nuclei and measurement of morphology and intensities.
  3. Sequential scatter plots: Size- and intensity-derived proxies separated G0/G1, S/G2, and mitotic populations.
  4. Mitotic isolation: A final gate on Major vs. Minor Axis Length isolated elongated, dividing cells.
Cytely interactive analysis interface
Figure 2: Cytely interactive analysis interface. The workflow uses a sequence of scatter plots for analysis. An initial plot (left) isolates healthy single cells. A second plot of Mean DAPI Intensity vs. Area (middle) separates interphase cells from a candidate mitotic population. A final plot of Major vs. Minor Axis Length (right) refines the selection to isolate elongated, dividing cells, with corresponding cropped images shown below for immediate visual verification.

Results

Three major populations were classified by morphology with preserved spatial context. Representative crops of gated cells confirm phenotype by visual inspection. Quantification across the dataset:

  • G0/G1: 65%
  • S/G2: 23%
  • Mitosis: 3%
  • Other: 9%
Isolation of the pure mitotic cell population
Figure: Isolation of the pure mitotic cell population. The scatter plot shows the final gate applied to cells based on their Major vs. Minor Axis Length. The cropped images below show only the selected cells, confirming their elongated nuclear morphology characteristic of metaphase and anaphase. (A) Mitotic cells showing condensed chromosomes and elongated nuclear morphology. (B) G0/G1 phase cells with small nuclei and lower DAPI intensity. (C) S/G2 phase cells with larger nuclei and higher DAPI intensity.

Conclusions

Cytely transforms cell cycle analysis into a fast, intuitive workflow. Automated segmentation combined with multi-parameter gating enables precise quantification of cell cycle stages using standard nuclear staining. The workflow preserves spatial context and provides immediate visual confirmation, yielding reproducible, publication-ready results.

  • Eliminates complex multi-dye protocols
  • Enables retrospective analysis of existing datasets
  • Preserves spatial context that flow cytometry loses
  • Immediate visual validation of gated populations