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Updated: Aug 8, 2026
Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry
Published on: May 9, 2019
Automatic classification of cells in cell cycle phases based on Ki-67 antigen quantification by fluorescence
P Guillaud1, J Vermont, D Seigneurin
1Equipe de Cytologie Quantitative, Faculté de Médecine de Grenoble, France.
Insights
This study introduces a novel method using Ki-67 staining and videomicrofluorometry to automatically classify cells into different cell cycle phases. This technique accurately quantifies cell proliferation and phase distribution, offering a faster alternative to existing methods.
Area of Science:
- Cell Biology
- Immunocytochemistry
- Flow Cytometry
Background:
- Ki-67 antigen is a widely used marker for cell proliferation, detected in cycling cells across G1, S, G2, and M phases.
- The immunocytochemical staining pattern of Ki-67 varies depending on the cell cycle phase, necessitating precise analytical methods.
- Existing methods for cell cycle analysis, such as DNA histograms and bromodeoxyuridine uptake, have limitations in speed and accuracy.
Purpose of the Study:
- To develop and validate a new method for automatic cell cycle phase classification using Ki-67 antigen staining.
- To identify optimal parameters from Ki-67 staining patterns for accurate cell classification.
- To compare the efficacy of this novel method with established techniques for proliferation assessment.
Main Methods:
- Utilized videomicrofluorometry for double staining of Ki-67 antigen and DNA in MCF-7 cell lines.
- Defined a set of parameters describing Ki-67 staining levels and patterns for individual cells.
- Applied statistical analyses, including principal component and discriminant analysis, to identify key classification parameters.
Main Results:
- Achieved a high cell classification rate of 94.37% across G1, S, G2, and M phases (prophase, metaphase, anaphase, telophase).
- Developed a program incorporating discriminant analysis coefficients for automatic calculation of Ki-67 labeling index and phase percentages.
- Demonstrated that the defined parameters effectively characterize cellular groups within different cell cycle stages.
Conclusions:
- The novel Ki-67 based method provides a rapid and accurate means for evaluating cell proliferation and cell cycle phase distribution.
- This approach offers a potentially more widely applicable alternative to traditional methods for cell cycle analysis.
- Automatic classification of cells based on Ki-67 staining patterns enhances efficiency in proliferation studies.
Abstract:
Ki-67 antigen is thought to be a marker of cell proliferation, as it can be detected in cycling cells, i.e. cells in G1, S, G2 and M phases, but not in resting cells. The immunocytochemical staining pattern obtained by the Ki-67 monoclonal antibody varies, depending on the cell cycle phases. Analysis of double staining of Ki-67 antigen and DNA in the MCF-7 cell line by videomicrofluorometry allows the description of both the level and the pattern of Ki-67 staining in the form of a set of parameters defining each cell. These parameters were measured in MCF-7 cell populations characterized according to their position in the cell cycle. They were submitted to a statistical analysis (principal component and discriminant analysis) which allowed the determination of the optimal parameters to characterize a given cellular group and permitted the use of these parameters for an automatic classification of cells in the different cell cycle phases. In G1, S, G2, prophase + metaphase and anaphase + telophase cells, these parameters allowed a classification of cells with a good-classification rate of 94.37%. A comparison of this method with methods based on the DNA histogram and bromodeoxyuridine uptake was performed. The classification coefficients stemming from the discriminant analysis were introduced into a program to obtain, automatically, the Ki-67 labelling index and the percentages of cells in each phase. This method, which allows a quick evaluation of the proliferation and the phase indices, may be more widely applicable.
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