Cell Cycle Analysis and Relevance for Single-Cell Gating in Mass Cytometry

Area of Science:

• Biomedical Sciences
• Cell Biology
• Analytical Chemistry

Background:

• Mass cytometry (MC) offers high-dimensional analysis but faces challenges in cell cycle profiling due to DNA intercalator resolution.
• Accurate cell cycle phase determination is crucial for understanding cellular processes and disease states.

Purpose of the Study:

• To develop and validate a robust cell cycle analysis panel for mass cytometry.
• To establish an analysis pipeline for true single-cell resolution in MC-based cell cycle studies.

Main Methods:

• A minimum cell cycle panel for MC was designed using an Iridium-labeled DNA intercalator, IdU, and antibodies against pS28HistoneH3, CDT1, and Geminin.
• An analysis pipeline incorporating unsupervised clustering (FlowSOM) and single-cell gating was developed to address cell aggregation issues in MC.
• The MC cell cycle panel and pipeline were validated against fluorescence flow cytometry, showing high correlation.

Main Results:

• The developed MC cell cycle panel achieved cell cycle distributions comparable to fluorescence flow cytometry (r² = 0.98).
• The analysis pipeline effectively identified and removed debris, dead cells, and non-single-cell aggregates.
• The panel allows for further subdivision of G1 and G2 phases using additional markers like anti-pS780RB1 and anti-PLK1.

Conclusions:

• The presented mass cytometry cell cycle panel and analysis pipeline provide a reliable method for high-resolution cell cycle analysis.
• This approach enables true single-cell analysis, preserving all channels for concurrent phenotyping and expression studies.
• The validated method enhances the utility of mass cytometry for comprehensive cell cycle research.